Removable plant transgenic loci with cognate guide rna recognition sites

ABSTRACT

Transgenic plants comprising an originator guide RNA recognition site (OgRRS) as well as an exogenous cognate guide RNA recognition site (CgRRS) which is introduced at or near the junctions of the transgene insert, methods of making such plants, and use of such plants to facilitate breeding are disclosed.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing contained in the file named “10083WO1_ST25.txt,”which is 492,562 bytes as measured in the Windows operating system, andwhich was created on Jul. 17, 2021 and electronically filed via EFS-Webon Jul. 26, 2021, is incorporated herein by reference in its entirety.

BACKGROUND

Transgenes which are placed into different positions in the plant genomethrough non-site specific integration can exhibit different levels ofexpression (Weising et al., 1988, Ann. Rev. Genet. 22:421-477). Suchtransgene insertion sites can also contain various undesirablerearrangements of the foreign DNA elements that include deletions and/orduplications. Furthermore, many transgene insertion sites can alsocomprise selectable or scoreable marker genes which in some instancesare no longer required once a transgenic plant event containing thelinked transgenes which confer desirable traits are selected.

Commercial transgenic plants typically comprise one or more independentinsertions of transgenes at specific locations in the host plant genomethat have been selected for features that include expression of thetransgene(s) of interest and the transgene-conferred trait(s), absenceor minimization of rearrangements, and normal Mendelian transmission ofthe trait(s) to progeny. Examples of selected transgenic corn, soybean,cotton, and canola plant events which confer traits such as herbicidetolerance and/or pest tolerance are disclosed in U.S. Pat. Nos.7,323,556; 8,575,434; 6,040,497; 10316330; 8618358; 8212113; 9428765;8455720; 7897748; 8273959; 8093453; 8901378; 8466346; RE44962; 9540655;9738904; 8680363; 8049071; 9447428; 9944945; 8592650; 10184134; 7179965;7371940; 9133473; 8735661; 7381861; 8048632; and 9738903.

Methods for removing selectable marker genes and/or duplicatedtransgenes in transgene insertion sites in plant genomes involving useof site-specific recombinase systems (e.g., cre-lox) as well as forinsertion of new genes into transgene insertion sites have beendisclosed (Srivastava and Ow; Methods Mol Biol, 2015,1287:95-103; Daleand Ow, 1991, Proc. Natl Acad. Sci. USA 88, 10558-10562; Srivastava andThomson, Plant Biotechnol J, 2016; 14(2):471-82). Such methods typicallyrequire incorporation of the recombination site sequences recognized bythe recombinase at particular locations within the transgene.

SUMMARY

Edited transgenic plant genomes comprising a first modified transgeniclocus that comprises: (i) a first originator guide RNA recognition site(OgRRS) comprising a protospacer adjacent motif (PAM) site operablylinked to a guide RNA hybridization site, wherein the OgRRS is locatedin non-transgenic plant genomic DNA of a first DNA junctionpolynucleotide of the first modified transgenic locus; and (ii) a firstcognate guide RNA recognition site (CgRRS) comprising a protospaceradjacent motif (PAM) site operably linked to a guide RNA hybridizationsite located in a second DNA junction polynucleotide of the firstmodified transgenic locus, wherein the CgRRS is absent from transgenicplant genomes comprising a first original transgenic locus that isunmodified and wherein the OgRRS and the CgRRS can hybridize to onefirst guide RNA (gRNA) are provided.

Transgenic plant cells, transgenic plants, and transgenic plant patsincluding a seed, leaf, tuber, stem, root, or boll that comprise theedited transgenic plant genome are also provided.

Methods of obtaining a plant breeding line comprising (a) crossing twotransgenic plants comprising the aforementioned edited transgenicgenomes comprising an OgRRS and a CgRRS, wherein a first plantcomprising the first modified transgenic locus is crossed to a secondplant comprising the second modified transgenic locus; and (b) selectinga progeny plant comprising the first and second modified transgeniclocus from the cross are provided. Methods for obtaining a bulkedpopulation of inbred seed for commercial seed production comprisingselfing the transgenic plants comprising the aforementioned editedtransgenic genomes comprising an OgRRS and a CgRRS and harvesting seedfrom the selfed plants are also provided. Methods of obtaining hybridcrop seed comprising crossing a first crop plant comprising thetransgenic plants comprising the aforementioned edited transgenicgenomes comprising an OgRRS and a CgRRS to a second crop plant andharvesting seed from the cross are provided.

DNA comprising a cognate guide RNA recognition site (CgRRS) and at least10 bp of transgenic DNA or non-transgenic plant genomic DNA flanking theCgRRS, wherein the transgenic DNA or non-transgenic plant genomic DNAcomprise transgenic or non-transgenic plant genomic DNA sequences of thesecond DNA junction polynucleotide of the first modified transgeniclocus is provided. Processed transgenic plant products containing theaforementioned DNA and biological samples containing the aforementionedDNA are also provided.

Nucleic acid markers adapted for detection of genomic DNA or fragmentsthereof comprising a cognate guide RNA recognition site (CgRRS) in,adjacent to, or operably linked to a DNA junction polynucleotide of amodified transgenic locus are provided.

Methods of detecting the edited transgenic plant genomes comprising thestep of detecting the presence of a polynucleotide comprising one ormore of the CgRRS are provided.

Methods of obtaining an edited transgenic plant genome comprising amodified transgenic locus comprising the step of introducing a cognateguide RNA recognition site (CgRRS) in a DNA junction polynucleotide ofan original transgenic locus, wherein the CgRRS is in, adjacent to, oroperably linked to a DNA junction polynucleotide of the modifiedtransgenic locus, are provided.

Methods of excising a modified transgenic locus from an editedtransgenic plant genome comprising the steps of: (a) contacting any ofthe aforementioned edited transgenic plant genome(s) with: (i) an RNAdependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capableof hybridizing to the guide RNA hybridization site of the first OgRRSand the first CgRRS; wherein the RdDe recognizes a OgRRS/gRNA and aCgRRS/gRNA hybridization complex; and, (b) selecting a transgenic plantcell, transgenic plant part, or transgenic plant wherein the modifiedtransgenic locus flanked by the first OgRRS and the first CgRRS has beenexcised are provided.

Methods for obtaining inbred transgenic plant germplasm containingdifferent transgenic traits comprising: (a) introgressing at least afirst transgenic locus and a second transgenic locus into inbredgermplasm to obtain a donor inbred parent plant line comprising thefirst and second transgenic loci, wherein a first OgRRS and a firstCgRRS are operably linked to both DNA junction polynucleotides of atleast the first transgenic locus and optionally wherein a second OgRRSand a second CgRRS are operably linked to the second transgenic locus;(b) contacting the transgenic plant genome of the donor inbred parentplant line with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii)a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridizationsite of the first OgRRS and the second CgRRS; wherein the RdDerecognizes a first OgRRS/gRNA and a first CgRRS/gRNA hybridizationcomplex; and (c) selecting a transgenic plant cell, transgenic plantpart, or transgenic plant comprising an edited transgenic plant genomein the inbred germplasm, wherein the first transgenic locus has beenexcised and the second transgenic locus is present in the inbredgermplasm are provided.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows a diagram of transgene expression cassettes and selectablemarkers in the DAS-59122-7 transgenic locus set forth in SEQ ID NO: 1.

FIG. 2 shows a diagram of transgene expression cassettes and selectablemarkers in the DP-4114 transgenic locus set forth in SEQ ID NO: 2.

FIG. 3 shows a diagram of transgene expression cassettes and selectablemarkers in the MON87411 transgenic locus.

FIG. 4 shows a diagram of transgene expression cassettes in the MON89034transgenic locus.

FIG. 5 shows a diagram of transgene expression cassettes and selectablemarkers in the MIR162 transgenic locus.

FIG. 6 shows a diagram of transgene expression cassettes and selectablemarkers in the MIR604 transgenic locus set forth in SEQ ID NO: 6.

FIG. 7 shows a diagram of transgene expression cassettes and selectablemarkers in the NK603 transgenic locus set forth in SEQ ID NO: 7.

FIG. 8 shows a diagram of transgene expression cassettes and selectablemarkers in the SYN-E3272-5 transgenic locus set forth in SEQ ID NO: 8.

FIG. 9 shows a diagram of transgene expression cassettes and selectablemarkers in the TC1507 transgenic locus set forth in SEQ ID NO: 8.

FIG. 10 shows a diagram of transgene expression cassettes and selectablemarkers in the 5307 transgenic locus set forth in SEQ ID NO: 10.

FIG. 11 shows a schematic diagram which compares current breedingstrategies for introgression of transgenic events (i.e., transgenicloci) to alternative breeding strategies for introgression of transgenicevents where the transgenic events (i.e., transgenic loci) can beremoved following introgression to provide different combinations oftransgenic traits. In FIG. 11 , “GE” refers to genome editing (e.g.,including introduction of targeted genetic changes with genome editingmolecules and “Event Removal” refers to excision of a transgenic locus(i.e., an “Event”) or portion thereof with genome editing molecules.

FIG. 12 shows a diagram of transgene expression cassettes and selectablemarkers in the DAS68416-4 transgenic locus set forth in SEQ ID NO: 12.

FIG. 13 shows a diagram of transgene expression cassettes and selectablemarkers in the MON87701 transgenic locus set forth in SEQ ID NO: 14.

FIG. 14 shows a diagram of transgene expression cassettes and selectablemarkers in the MON89788 transgenic locus set forth in SEQ ID NO: 16.

FIG. 15 shows a diagram of transgene expression cassettes and selectablemarkers in the COT102 transgenic locus set forth in SEQ ID NO: 19.

FIG. 16 shows a diagram of transgene expression cassettes and selectablemarkers in the MON88302 transgenic locus set forth in SEQ ID NO: 21.

FIG. 17A, B, C. FIG. 17A shows a schematic diagram of a non-limitingexample of: (i) an untransformed plant chromosome containingnon-transgenic DNA which includes the originator guide RNA recognitionsite (OgRRS) (top); (ii) the original transgenic locus with the OgRRS inthe non-transgenic DNA of the 1st junction polynucleotide (middle); and(iii) the modified transgenic locus with a cognate guide RNA insertedinto the non-transgenic DNA of the 2nd junction polynucleotide (bottom).FIG. 17B shows a schematic diagram of a non-limiting example of aprocess where a modified transgenic locus with a cognate guide RNAinserted into the non-transgenic DNA of the 2nd junction polynucleotide(top) is subjected to cleavage at the OgRRS and CgRRS with one guide RNA(gRNA) that hybridizes to gRNA hybridization site in both the OgRRS andthe CgRRS and an RNA dependent DNA endonuclease (RdDe) that recognizesand cleaves the gRNA/OgRRS and the gRNA/CgRRS complex followed bynon-homologous end joining processes to provide a plant chromosome wherethe transgenic locus is excised. FIG. 17C shows a schematic diagram of anon-limiting example of a process where a modified transgenic locus witha cognate guide RNA inserted into the non-transgenic DNA of the 2ndjunction polynucleotide (top) is subjected to cleavage at the OgRRS andCgRRS with one guide RNA (gRNA) that hybridizes to the gRNAhybridization site in both the OgRRS and the CgRRS and an RNA dependentDNA endonuclease (RdDe) that recognizes and cleaves the gRNA/OgRRS andthe gRNA/CgRRS complex in the presence of a donor DNA template. In FIG.17C, cleavage of the modified transgenic locus in the presence of thedonor DNA template which has homology to non-transgenic DNA but lacksthe OgRRS in the 1st and 2nd junction polynucleotides followed byhomology-directed repair processes to provide a plant chromosome wherethe transgenic locus is excised and non-transgenic DNA present in theuntransformed plant chromosome is at least partially restored.

DETAILED DESCRIPTION

Unless otherwise stated, nucleic acid sequences in the text of thisspecification are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA or as RNA, asspecified; disclosure of one necessarily defines the other, as well asnecessarily defines the exact complements, as is known to one ofordinary skill in the art.

Where a term is provided in the singular, the inventors also contemplateembodiments described by the plural of that term.

The term “about” as used herein means a value or range of values whichwould be understood as an equivalent of a stated value and can begreater or lesser than the value or range of values stated by 10percent. Each value or range of values preceded by the term “about” isalso intended to encompass the embodiment of the stated absolute valueor range of values.

The phrase “allelic variant” as used herein refers to a polynucleotideor polypeptide sequence variant that occurs in a different strain,variety, or isolate of a given organism.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the phrase “approved transgenic locus” is a geneticallymodified plant event which has been authorized, approved, and/orde-regulated for any one of field testing, cultivation, humanconsumption, animal consumption, and/or import by a governmental body.Illustrative and non-limiting examples of governmental bodies whichprovide such approvals include the Ministry of Agriculture of Argentina,Food Standards Australia New Zealand, National Biosafety TechnicalCommittee (CTNBio) of Brazil, Canadian Food Inspection Agency, ChinaMinistry of Agriculture Biosafety Network, European Food SafetyAuthority, US Department of Agriculture, US Department of EnvironmentalProtection, and US Food and Drug Administration.

The term “backcross”, as used herein, refers to crossing an F1 plant orplants with one of the original parents. A backcross is used to maintainor establish the identity of one parent (species) and to incorporate aparticular trait from a second parent (species). The term “backcrossgeneration”, as used herein, refers to the offspring of a backcross.

As used herein, the phrase “biological sample” refers to either intactor non-intact (e.g. milled seed or plant tissue, chopped plant tissue,lyophilized tissue) plant tissue. It may also be an extract comprisingintact or non-intact seed or plant tissue. The biological sample cancomprise flour, meal, syrup, oil, starch, and cereals manufactured inwhole or in part to contain crop plant by-products. In certainembodiments, the biological sample is “non-regenerable” (i.e., incapableof being regenerated into a plant or plant part). In certainembodiments, the biological sample refers to a homogenate, an extract,or any fraction thereof containing genomic DNA of the organism fromwhich the biological sample was obtained, wherein the biological sampledoes not comprise living cells.

As used herein, the terms “correspond,” “corresponding,” and the like,when used in the context of an nucleotide position, mutation, and/orsubstitution in any given polynucleotide (e.g., an allelic variant ofSEQ ID NO: 1-34) with respect to the reference polynucleotide sequence(e.g., SEQ ID NO: 1-34) all refer to the position of the polynucleotideresidue in the given sequence that has identity to the residue in thereference nucleotide sequence when the given polynucleotide is alignedto the reference polynucleotide sequence using a pairwise alignmentalgorithm (e.g., CLUSTAL O 1.2.4 with default parameters).

As used herein, the terms “Cpf1” and “Cas12a” are used interchangeablyto refer to the same RNA dependent DNA endonuclease (RdDe). Cas12aproteins include the protein provided herein as SEQ ID NO: 46.

The term “crossing” as used herein refers to the fertilization of femaleplants (or gametes) by male plants (or gametes). The term “gamete”refers to the haploid reproductive cell (egg or pollen) produced inplants by meiosis from a gametophyte and involved in sexualreproduction, during which two gametes of opposite sex fuse to form adiploid zygote. The term generally includes reference to a pollen(including the sperm cell) and an ovule (including the ovum). Whenreferring to crossing in the context of achieving the introgression of agenomic region or segment, the skilled person will understand that inorder to achieve the introgression of only a part of a chromosome of oneplant into the chromosome of another plant, random portions of thegenomes of both parental lines recombine during the cross due to theoccurrence of crossing-over events in the production of the gametes inthe parent lines. Therefore, the genomes of both parents must becombined in a single cell by a cross, where after the production ofgametes from the cell and their fusion in fertilization will result inan introgression event.

As used herein, the phrases “DNA junction polynucleotide” and “junctionpolynucleotide” refers to a polynucleotide of about 18 to about 500 basepairs in length comprised of both endogenous chromosomal DNA of theplant genome and heterologous transgenic DNA which is inserted in theplant genome. A junction polynucleotide can thus comprise about 8, 10,20, 50, 100, 200, 250, 500, or 1000 base pairs of endogenous chromosomalDNA of the plant genome and about 8, 10, 20, 50, 100, 200, 250, 500, or1000 base pairs of heterologous transgenic DNA which span the one end ofthe transgene insertion site in the plant chromosomal DNA. Transgeneinsertion sites in chromosomes will typically contain both a 5′ junctionpolynucleotide and a 3′ junction polynucleotide. In embodiments setforth herein in SEQ ID NO: 1-34, the 5′ junction polynucleotide islocated at the 5′ end of the sequence and the 3′ junction polynucleotideis located at the 3′ end of the sequence. In a non-limiting andillustrative example, a 5′ junction polynucleotide of a transgenic locusis telomere proximal in a chromosome arm and the 3′ junctionpolynucleotide of the transgenic locus is centromere proximal in thesame chromosome arm. In another non-limiting and illustrative example, a5′ junction polynucleotide of a transgenic locus is centromere proximalin a chromosome arm and the 3′ junction polynucleotide of the transgeniclocus is telomere proximal in the same chromosome arm. The junctionpolynucleotide which is telomere proximal and the junctionpolynucleotide which is centromere proximal can be determined bycomparing non-transgenic genomic sequence of a sequenced non-transgenicplant genome to the non-transgenic DNA in the junction polynucleotides.

The term “donor,” as used herein in the context of a plant, refers tothe plant or plant line from which the trait, transgenic event, orgenomic segment originates, wherein the donor can have the trait,introgression, or genomic segment in either a heterozygous or homozygousstate.

As used herein, the terms “excise” and “delete,” when used in thecontext of a DNA molecule, are used interchangeably to refer to theremoval of a given DNA segment or element (e.g., transgene element ortransgenic locus) of the DNA molecule.

As used herein, the phrase “elite crop plant” refers to a plant whichhas undergone breeding to provide one or more trait improvements. Elitecrop plant lines include plants which are an essentially homozygous,e.g. inbred or doubled haploid. Elite crop plants can include inbredlines used as is or used as pollen donors or pollen recipients in hybridseed production (e.g. used to produce F1 plants). Elite crop plants caninclude inbred lines which are selfed to produce non-hybrid cultivars orvarieties or to produce (e.g., bulk up) pollen donor or recipient linesfor hybrid seed production. Elite crop plants can include hybrid F1progeny of a cross between two distinct elite inbred or doubled haploidplant lines.

As used herein, an “event,” “a transgenic event,” “a transgenic locus”and related phrases refer to an insertion of one or more transgenes at aunique site in the genome of a plant as well as to DNA fragments, plantcells, plants, and plant parts (e.g., seeds) comprising genomic DNAcontaining the transgene insertion. Such events typically comprise botha 5′ and a 3′ DNA junction polynucleotide and confer one or more usefultraits including herbicide tolerance, insect resistance, male sterility,and the like.

As used herein, the phrases “endogenous sequence,” “endogenous gene,”“endogenous DNA,” “endogenous polynucleotide,” and the like refer to thenative form of a polynucleotide, gene or polypeptide in its naturallocation in the organism or in the genome of an organism.

The terms “exogenous” and “heterologous” as are used synonymously hereinto refer to any polynucleotide (e.g. DNA molecule) that has beeninserted into a new location in the genome of a plant. Non-limitingexamples of an exogenous or heterologous DNA molecule include asynthetic DNA molecule, a non-naturally occurring DNA molecule, a DNAmolecule found in another species, a DNA molecule found in a differentlocation in the same species, and/or a DNA molecule found in the samestrain or isolate of a species, where the DNA molecule has been insertedinto a new location in the genome of a plant.

As used herein, the term “F1” refers to any offspring of a cross betweentwo genetically unlike individuals.

The term “gene,” as used herein, refers to a hereditary unit consistingof a sequence of DNA that occupies a specific location on a chromosomeand that contains the genetic instruction for a particularcharacteristics or trait in an organism. The term “gene” thus includes anucleic acid (for example, DNA or RNA) sequence that comprises codingsequences necessary for the production of an RNA, or a polypeptide orits precursor. A functional polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence as long as thedesired activity or functional properties (e.g., enzymatic activity,pesticidal activity, ligand binding, and/or signal transduction) of theRNA or polypeptide are retained.

The term “identifying,” as used herein with respect to a plant, refersto a process of establishing the identity or distinguishing character ofa plant, including exhibiting a certain trait, containing one or moretransgenes, and/or containing one or more molecular markers.

The term “isolated” as used herein means having been removed from itsnatural environment.

As used herein, the terms “include,” “includes,” and “including” are tobe construed as at least having the features to which they refer whilenot excluding any additional unspecified features.

As used herein, the phrase “introduced transgene” is a transgene notpresent in the original transgenic locus in the genome of an initialtransgenic event or in the genome of a progeny line obtained from theinitial transgenic event. Examples of introduced transgenes includeexogenous transgenes which are inserted in a resident originaltransgenic locus.

As used herein, the terms “introgression,” “introgressed,” and“introgressing” refer to both a natural and artificial process, and theresulting plants, whereby traits, genes or DNA sequences of one species,variety or cultivar are moved into the genome of another species,variety or cultivar, by crossing those species. The process mayoptionally be completed by backcrossing to the recurrent parent.Examples of introgression include entry or introduction of a gene, atransgene, a regulatory element, a marker, a trait, a trait locus, or achromosomal segment from the genome of one plant into the genome ofanother plant.

The phrase “marker-assisted selection,” as used herein, refers to thediagnostic process of identifying, optionally followed by selecting aplant from a group of plants using the presence of a molecular marker asthe diagnostic characteristic or selection criterion. The processusually involves detecting the presence of a certain nucleic acidsequence or polymorphism in the genome of a plant.

The phrase “molecular marker,” as used herein, refers to an indicatorthat is used in methods for visualizing differences in characteristicsof nucleic acid sequences. Examples of such indicators are restrictionfragment length polymorphism (RFLP) markers, amplified fragment lengthpolymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs),microsatellite markers (e.g. SSRs), sequence-characterized amplifiedregion (SCAR) markers, Next Generation Sequencing (NGS) of a molecularmarker, cleaved amplified polymorphic sequence (CAPS) markers or isozymemarkers or combinations of the markers described herein which defines aspecific genetic and chromosomal location.

As used herein the terms “native” or “natural” define a condition foundin nature. A “native DNA sequence” is a DNA sequence present in naturethat was produced by natural means or traditional breeding techniquesbut not generated by genetic engineering (e.g., using molecularbiology/transformation techniques).

The term “offspring”, as used herein, refers to any progeny generationresulting from crossing, selfing, or other propagation technique.

The phrase “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression. When the phrase “operably linked” is used in the context ofa PAM site and a guide RNA hybridization site, it refers to a PAM sitewhich permits cleavage of at least one strand of DNA in a polynucleotidewith an RNA dependent DNA endonuclease or RNA dependent DNA nickasewhich recognize the PAM site when a guide RNA complementary to guide RNAhybridization site sequences adjacent to the PAM site is present. AOgRRS and its CgRRS are operably linked to junction polynucleotides whenthey can be recognized by a gRNA and an RdDe to provide for excision ofthe transgenic locus or portion thereof flanked by the junctionpolynucleotides.

As used herein, the term “plant” includes a whole plant and anydescendant, cell, tissue, or part of a plant. The term “plant parts”include any part(s) of a plant, including, for example and withoutlimitation: seed (including mature seed and immature seed); a plantcutting; a plant cell; a plant cell culture; or a plant organ (e.g.,pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, andexplants). A plant tissue or plant organ may be a seed, protoplast,callus, or any other group of plant cells that is organized into astructural or functional unit. A plant cell or tissue culture may becapable of regenerating a plant having the physiological andmorphological characteristics of the plant from which the cell or tissuewas obtained, and of regenerating a plant having substantially the samegenotype as the plant. Regenerable cells in a plant cell or tissueculture may be embryos, protoplasts, meristematic cells, callus, pollen,leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs,husks, or stalks. In contrast, some plant cells are not capable of beingregenerated to produce plants and are referred to herein as“non-regenerable” plant cells.

The term “purified,” as used herein defines an isolation of a moleculeor compound in a form that is substantially free of contaminantsnormally associated with the molecule or compound in a native or naturalenvironment and means having been increased in purity as a result ofbeing separated from other components of the original composition. Theterm “purified nucleic acid” is used herein to describe a nucleic acidsequence which has been separated from other compounds including, butnot limited to polypeptides, lipids and carbohydrates.

The term “recipient”, as used herein, refers to the plant or plant linereceiving the trait, transgenic event or genomic segment from a donor,and which recipient may or may not have the have trait, transgenic eventor genomic segment itself either in a heterozygous or homozygous state.

As used herein the term “recurrent parent” or “recurrent plant”describes an elite line that is the recipient plant line in a cross andwhich will be used as the parent line for successive backcrosses toproduce the final desired line.

As used herein the term “recurrent parent percentage” relates to thepercentage that a backcross progeny plant is identical to the recurrentparent plant used in the backcross. The percent identity to therecurrent parent can be determined experimentally by measuring geneticmarkers such as SNPs and/or RFLPs or can be calculated theoreticallybased on a mathematical formula.

The terms “selfed,” “selfing,” and “self,” as used herein, refer to anyprocess used to obtain progeny from the same plant or plant line as wellas to plants resulting from the process. As used herein, the terms thusinclude any fertilization process wherein both the ovule and pollen arefrom the same plant or plant line and plants resulting therefrom.

Typically, the terms refer to self-pollination processes and progenyplants resulting from self-pollination.

The term “selecting”, as used herein, refers to a process of picking outa certain individual plant from a group of individuals, usually based ona certain identity, trait, characteristic, and/or molecular marker ofthat individual.

As used herein, the phrase “originator guide RNA recognition site” orthe acronym “OgRRS” refers to an endogenous or exogenous DNApolynucleotide comprising a protospacer adjacent motif (PAM) siteoperably linked to a guide RNA hybridization site. In certainembodiments, an OgRRS can be located in an untransformed plantchromosome or in non-transgenic DNA of a DNA junction polynucleotide ofboth an original transgenic locus and a modified transgenic locus. Incertain embodiments, an OgRRS can be located in transgenic DNA of a DNAjunction polynucleotide of both an original transgenic locus and amodified transgenic locus. In certain embodiments, an OgRRS can belocated in both transgenic DNA and non-transgenic DNA of a DNA junctionpolynucleotide of both an original transgenic locus and a modifiedtransgenic locus (i.e., can span transgenic and non-transgenic DNA in aDNA junction polynucleotide).

As used herein the phrase “cognate guide RNA recognition site” or theacronym “CgRRS” refer to a DNA polynucleotide comprising a PAM siteoperably linked to a guide RNA hybridization site, where the CgRRS isabsent from transgenic plant genomes comprising a first originaltransgenic locus that is unmodified and where the CgRRS and itscorresponding OgRRS can hybridize to a single gRNA. A CgRRS can belocated in transgenic DNA of a DNA junction polynucleotide of a modifiedtransgenic locus, in transgenic DNA of a DNA junction polynucleotide ofa modified transgenic locus, or in both transgenic and non-transgenicDNA of a modified transgenic locus (i.e., can span transgenic andnon-transgenic DNA in a DNA junction polynucleotide).

As used herein, the phrase “a transgenic locus excision site” refers tothe DNA which remains in the genome of a plant or in a DNA molecule(e.g., an isolated or purified DNA molecule) wherein a segmentcomprising, consisting essentially of, or consisting of a transgeniclocus or portion thereof has been deleted. In a non-limiting andillustrative example, a transgenic locus excision site can thus comprisea contiguous segment of DNA comprising at least 10 base pairs of DNAthat is telomere proximal to the deleted transgenic locus or to thedeleted segment of the transgenic locus and at least 10 base pairs ofDNA that is centromere proximal to the deleted transgenic locus or tothe deleted segment of the transgenic locus.

As used herein, the phrase “transgene element” refers to a segment ofDNA comprising, consisting essentially of, or consisting of a promoter,a 5′ UTR, an intron, a coding region, a 3′UTR, or a polyadenylationsignal. Polyadenylation signals include transgene elements referred toas “terminators” (e.g., NOS, pinII, rbcs, Hsp17, TubA).

To the extent to which any of the preceding definitions is inconsistentwith definitions provided in any patent or non-patent referenceincorporated herein by reference, any patent or non-patent referencecited herein, or in any patent or non-patent reference found elsewhere,it is understood that the preceding definition will be used herein.

Genome editing molecules can permit introduction of targeted geneticchange conferring desirable traits in a variety of crop plants (Zhang etal. Genome Biol. 2018; 19: 210; Schindele et al. FEBS Lett. 2018;592(12):1954). Desirable traits introduced into crop plants such asmaize and soybean include herbicide tolerance, improved food and/or feedcharacteristics, male-sterility, and drought stress tolerance.Nonetheless, full realization of the potential of genome editing methodsfor crop improvement will entail efficient incorporation of the targetedgenetic changes in germplasm of different elite crop plants adapted fordistinct growing conditions. Such elite crop plants will also desirablycomprise useful transgenic loci which confer various traits includingherbicide tolerance, pest resistance (e.g.; insect, nematode, fungaldisease, and bacterial disease resistance), conditional male sterilitysystems for hybrid seed production, abiotic stress tolerance (e.g.,drought tolerance), improved food and/or feed quality, and improvedindustrial use (e.g., biofuel). Provided herein are methods wherebytargeted genetic changes are efficiently combined with desired subsetsof transgenic loci in elite progeny plant lines (e.g., elite inbredsused for hybrid seed production or for inbred varietal production). Alsoprovided are plant genomes containing modified transgenic loci which canbe selectively excised with a single gRNA molecule. Such modifiedtransgenic loci comprise an originator guide RNA recognition site(OgRRS) which is identified in non-transgenic DNA of a first junctionpolynucleotide of the transgenic locus and cognate guide RNA recognitionsite (CgRRS) which is introduced (e.g., by genome editing methods) intoa second junction polynucleotide of the transgenic locus and which canhybridize to the same gRNA as the OgRRS, thereby permitting excision ofthe modified transgenic locus or portion thereof with a single guideRNA. Use of a single guide RNA molecule rather than two guide RNAmolecules to excise a transgenic locus can be advantageous. In certainembodiments, the frequency of excision of the modified transgenic locusor a portion thereof comprising the OgRRS and CgRRS in the transgenicplant cells, calli, transgenic plant parts, or transgenic plantssubjected to treatment with the same guide RNA and an RdDe is greaterthan the frequency of excision of the modified transgenic locus or aportion thereof in control transgenic plant cells, calli, transgenicplant parts, or transgenic plants treated with two control gRNAs (e.g.,two distinct gRNAs that hybridize to DNA flanking all or most of themodified transgenic locus or a portion thereof) and an RdDe. Suchfrequencies of excision can be determined by treating equivalent numbersof plant cells, calli, plant parts, or plants with either the singlegRNA that hybridizes to the OgRRS and CgRRS plus an RdDe or with the twocontrol gRNAs plus an RdDe and determining the number of selected plantcells, calli, plant parts, or plants where the transgenic locus orportion thereof was excised. In certain embodiments, a portion of themodified transgenic locus is excised when the OgRRS, the CgRRS, or boththe OgRRS are located within transgenic DNA of the junction sequences.Since the CGRRS and/or OgRRS are typically located in or near the DNAjunction polynucleotides of the transgenic locus, portions of atransgenic locus that are excised typically comprise all or the bulk ofthe transgene expression cassettes (e.g., promoters which are operablylinked to genes which confer traits of interest and/or a selectablephenotype). In certain embodiments, an originator guide RNA recognitionsite (OgRRS) comprises endogenous DNA found in untransformed plants andin endogenous non-transgenic DNA of junction polynucleotides oftransgenic plants containing a modified or unmodified transgenic locus.In certain embodiments, an originator guide RNA recognition site (OgRRS)comprises exogenous transgenic DNA of junction polynucleotides oftransgenic plants containing a modified or unmodified transgenic locus.The OgRRS located in non-transgenic DNA, transgenic DNA, or acombination thereof in a first DNA junction polynucleotide is used todesign a related cognate guide RNA recognition site (CgRRS) which isintroduced (e.g., by genome editing methods) into the second junctionpolynucleotide of the transgenic locus. A CgRRS is thus present injunction polynucleotides of modified transgenic loci provided herein andis absent from endogenous DNA found in untransformed plants and absentfrom endogenous non-transgenic DNA found in junction sequences oftransgenic plants containing an unmodified transgenic locus. A CgRRS isalso absent from a combination of non-transgenic and transgenic DNAfound in junction sequences of transgenic plants containing anunmodified transgenic locus. Also provided are unique transgenic locusexcision sites created by excision of such modified transgenic loci, DNAmolecules comprising the modified transgenic loci, unique transgeniclocus excision sites and/or plants comprising the same, biologicalsamples containing the DNA, nucleic acid markers adapted for detectingthe DNA molecules, and related methods of identifying the elite cropplants comprising unique transgenic locus excision sites.

Further provided herein are improvements of pre-existing transgenic lociin plant genomes by directed insertion, deletion, and/or substitution ofDNA within or adjacent to such insertions as well as methods foreffecting and using such improvements. In certain embodiments, improvedtransgenic loci provided here are characterized by polynucleotidesequences that can facilitate as necessary the removal of the transgenicloci from the genome. Useful applications of such improved transgenicloci and related methods of making include targeted excision of a giventransgenic locus or portions thereof in certain breeding lines tofacilitate recovery of germplasm with subsets of transgenic traitstailored for specific geographic locations and/or grower preferences.Other useful applications of such improved transgenic loci and relatedmethods of making include removal of transgenic traits from certainbreeding lines when it is desirable to replace the trait in the breedingline without disrupting other transgenic loci and/or non-transgenicloci. In certain embodiments, the improved transgenic loci can providefor insertion of new transgenes that confer the replacement or otherdesirable trait at the genomic location of the improved transgeniclocus.

Methods provided herein can be used to excise any transgenic locus wherethe first and second junction sequences comprising the endogenousnon-transgenic genomic DNA and the heterologous transgenic DNA which arejoined at the site of transgene insertion in the plant genome are knownor have been determined. In certain embodiments provided herein,transgenic loci can be removed from crop plant lines to obtain cropplant lines with tailored combinations of transgenic loci and optionallytargeted genetic changes. Such first and second junction sequences arereadily identified in new transgenic events by inverse PCR techniquesusing primers which are complementary the inserted transgenic sequences.In certain embodiments, the first and second junction sequences oftransgenic loci are published. Examples of transgenic loci which can beimproved and used in the methods provided herein include the maize,soybean, cotton, and canola transgenic loci set forth in Tables 1, 2, 3,and 4, respectively. Transgenic junction sequences for certain eventsare also depicted in the drawings. Such transgenic loci set forth inTables 1-4 are found in crop plants which have in some instances beencultivated, been placed in commerce, and/or have been described in avariety of publications by various governmental bodies. Databases whichhave compiled descriptions of approved transgenic loci including theloci set forth in Tables 1-4 include the International Service for theAcquisition of Agri-biotech Applications (ISAAA) database (available onthe world wide web internet site “isaaa.org/gmapprovaldatabase/event”),the GenBit LLC database (available on the world wide web internet site“genbitgroup.com/en/gmo/gmodatabase”), and the Biosafety Clearing-House(BCH) database (available on the http internet site“_bch.cbd.int/database/organisms”).

TABLE 1 Corn Events (transgenic loci) Patent or ATCC or SEQ Event PatentNCIMB Trait ID NO Name Application Deposit expression (Figure (traits)¹Number(s)² Designation cassette(s) Number) BVLA430101 CN2013103194381AphyA2 (Q) Bt10 (IR, Cry1Ab, HT) PAT Bt11 (IR, U.S. Pat. No. 6,342,660;ATCC Cry1Ab and HT) U.S. Pat. No. 6,403,865; 209671 PAT U.S. Pat. No.6,943,282 Bt176 Cry1Ab, PAT CBH-351 JP PAT, Cry9c (HT, IR) 2006197926ADAS- U.S. Pat. No. 6,127,180; PTA-11384 cry34Ab1, SEQ ID 59122-7 U.S.Pat. No. 6,340,593; cry35Ab1, NO: 1 (IR, HT) U.S. Pat. No. 6,548,291;PAT (FIG. 1) U.S. Pat. No. 6,624,145; U.S. Pat. No. 6,893,872; U.S. Pat.No. 6,900,371; U.S. Pat. No. 7,323,556 (Event); U.S. Pat. No. 7,695,914(Event); U.S. Pat. No. 7,696,341; U.S. Pat. No. 7,956,246 (Event); U.S.Pat. No. 8,592,653 (Event); U.S. Pat. No. 8,952,223 (Event); RE 43,373;U.S. Pat. No. 9,878,321 (Event) DAS- US PTA-10244 aad-1 SEQ ID 40278(HT) 20120244533 NO: 22 DBT418 Cry1Ac, (IR, HT) PAT, pinII DP-4114 U.S.Pat. No. 8,575,434; PTA-11506 Cry1F, SEQ ID (IR, HT) U.S. Pat. No.10,190,179; cry34Ab1, NO: 2 US cry35Ab1, (FIG. 2) 20190136331 PATDP-32138 US PTA-9158 Zm Ms45, SEQ ID (MS, MSR) 20130031674 Zm aa1 NO: 24US gene, 20090038026 DsRed2 US 20060288440 DP-33121 US PTA-13392Cry2A.127, SEQ ID (IR. HT) 20150361446 Cry1A.88, NO: 23 VIP3Aa20, PATGA21 (HT) US ATCC EPSPS 2005086719; 209033 U.S. Pat. No. 6,040,497; U.S.Pat. No. 6,762,344; U.S. Pat. No. 7,314,970 HCEM485 U.S. Pat. No.8,759,618 PTA-12014 zmEPSPS SEQ ID (HT) B2 NO: 25 LY038 (Q) U.S. Pat.No. 7,157,281 PTA-5623 cordapA SEQ ID NO: 26 MON810 U.S. Pat. No.6,852,915 PTA-6260 Cry1Ab, (IR, HT, goxv247, AR) cp4epsps MON832Goxv247, (HT) cp4 epsps, nptII MON863 U.S. Pat. No. 7,705,216 PTA-2605Cry3Bb1 (IR) MON87403 US PTA-13584 athb17 SEQ ID (YG) 20170088904 NO: 27MON87411 U.S. Pat. No. 10,316,330 PTA-12669 cry3Bb1, SEQ ID (IR, HT)cp4epsps, NO: 3 dvsnf7 (FIG. 3) MON87419 US PTA- DMO, PAT SEQ ID (HT)2015/0267221 120860 NO: 28 MON87427 U.S. Pat. No. 8,618,358 PTA-7899cp4epsps (HT/MS)³ MON87460 U.S. Pat. No. 8,450,561 PTA-8910 cspB SEQ ID(AST) NO: 29 MON88017 U.S. Pat. No. 8,212,113; PTA-5582 cry3Bb1, (IR,HT) U.S. Pat. No. 8,686,230 cp4epsps MON89034 U.S. Pat. No. 9,428,765PTA-7455 cry2Ab2, SEQ ID (IR)⁴ cry1A.105 NO: 4 (FIG. 4) MIR162 U.S. Pat.No. 8,455,720 PTA-8166 VIP3Aa20 SEQ ID (IR, MU) NO: 5 (FIG. 5) MIR604U.S. Pat. No. 7,897,748 none cry3A055 SEQ ID (IR, MU) NO: 6 (FIG. 6) MS3Barnase, PAT MS6 barnase MZHG0JG US_201662346688_P PTA- ZmEPSPS, SEQ ID(HT) WO 122835 PAT NO: 30 2017214074 MZIR098 US PTA- ecry3.1Ab, SEQ ID(IR, HT) 20200190533 124143 mcry3A, NO: 31 PAT MYDT09Y DP-E29 NK603 U.S.Pat. No. 8,273,959 PTA-2478 cp4epsps SEQ ID (HT) NO: 7 (FIG. 7) SYN-U.S. Pat. No. 8,093,453 PTA-9972 amy797E SEQ ID E3272-5 NO: 8 (BF, MU)(FIG. 8) T14 (HT) PAT T25 (HT) PAT TC1507 U.S. Pat. No. 8,901,378;PTA-5448 cry1Fa2, SEQ ID (IR, HT) U.S. Pat. No. 8,502,047 (Inbred PATNO: 9 BE1146BMR); (Fig. 9) PTA-8519 (LLD06BM) TC6275 PAT, (IR, HT)moCry1F VCO- U.S. Pat. No. 9,994,863 NCIMB EPSPS SEQ ID Ø1981-5 41842NO: 32 (HT) 676 (MS, dam, PAT HT) 678 (MS, dam, PAT HT) 680 dam, PAT(MS, HT) 98140 (HT) U.S. Pat. No. 7,928,296 PTA-8296 zm-hra, SEQ ID GATNO: 33 5307 (IR, U.S. Pat. No. 8,466,346 PTA-9561 ecry3.1Ab SEQ ID MU)NO: 10 (FIG. 10) ¹Traits: IR = Insect Resistance; HT = HerbicideTolerance; AR = Antibiotic Resistance; MU = mannose utilization; BF =Biofuel; MS = Male Sterility; MSR = Male Sterility Restoration; Q = Foodand/or Feed Quality; AST = Abiotic Stress Tolerance; YG = Yield/Growth.²Each US Patent or Patent Application Publication is incorporated hereinby reference in its entirety. ³A single transgene confers vegetativetolerance to glyphosate and exhibits glyphosate-induced male sterility.⁴Resistance to coleopteran and lepidopteran insect pests.

TABLE 2 Soybean Events (transgenic loci) Patent or ATCC;³ or SEQ EventPatent NCIMB⁴ Trait ID NO Name Application Deposit expression (Figure(traits)¹ Number(s)² Number cassette(s) Number) A5547-127 US NCIMB PAT(HT) 20080196127 41660 RE44962 DAS44406- U.S. Pat. No. PTA-11336 Aad-12,SEQ ID 6 (HT)⁵ 9,540,655 2mepsps, NO: 11 U.S. Pat. No. PAT 10,400,250DAS68416- U.S. Pat. No. PTA-10442 Aad-12, SEQ ID 4 (IR, HT)⁶ 9,738,904PTA-12006 PAT NO: 12 (FIG. 12) DAS81419- U.S. Pat. No. PTA-12006 cry1Ac,SEQ ID 2 (IR, HT) 8,680,363 cry1F, PAT NO: 13 U.S. Pat. No. 8,632,978U.S. Pat. No. 9,695,441 U.S. Pat. No. 9,738,904 GTS 40-3-2 US M690GT |cp4epsps (HT) 20070136836 0.9 RM Soybean⁷ MON87701 U.S. Pat. No.PTA-8194 cry1Ac SEQ ID (IR) 8,049,071 NO: 14 (FIG. 13) MON87708 U.S.Pat. No. PTA-9670 DMO SEQ ID (HT)⁸ 9,447,428 NO: 15 MON89788 U.S. Pat.No. PTA-6708 cp4epsps SEQ ID (HT) 9,944,945 NO: 16 (FIG. 14) MST- U.S.Pat. No. NCIMB hppdPF SEQ ID FGØ72-3 8,592,650 41659 W336, NO: 34 (HT)⁹2mepsps SYHT0H2¹⁰ U.S. Pat. No. PTA-11226 cAvHPPD- 10,184,134 03¹Traits: IR = Insect Resistance; HT = Herbicide Tolerance; AR =Antibiotic Resistance; MU = mannose utilization; BF = Biofuel; MS = MaleSterility. ²Each US Patent or Patent Application Publication isincorporated herein by reference in its entirety. ³ATCC is the AmericanType Culture Collection, 10801 University Boulevard Manassas, VA 20110USA (for “PTA-XXXXX” deposits). ⁴NCIMB is the National Collection ofIndustrial, Food and Marine Bacteria, Ferguson Building, CraibstoneEstate, Bucksburn, Aberdeen AB9YA, Scotland. ⁵HT to 2,4-D; glyphosate,and glufosinate; also refered to as pDAB8264.44.06.1. ⁶Independent IR/HTand HT events combined by breeding. IR/HT event (Cry1F, Cry1Ac synpro(Cry1Ac), and PAT) is DAS81419-2, deposited with ATCC under PTA-12006,also referred to as DAS81419-2. ⁷ Elk Mound Seed, 308 Railroad StreetElk Mound, WI, USA 54739. ⁸HT to dicamba. ⁹HT to both glyphosate andisoxaflutole herbicides. ¹⁰HT to glufosinate and mesotrione herbicides.

TABLE 3 Cotton Events (transgenic loci) SEQ Trait ID NO Event NamePatent ATCC expression (Figure (traits) ¹ Number Deposit cassette(s)Number) DAS-21023- U.S. Pat. No. PTA-6233 Cry1Ac, SEQ ID 5 (IR, HT) ¹7,179,965 PAT NO: 17 DAS-24236- U.S. Pat. No. PTA-6233 Cry1F, SEQ ID5(IR, HT) ¹ 7,179,965 PAT NO: 18 COT102 (IR, U.S. Pat. No. Vip3A(a), SEQID AR) ² 7,371,940 NO: 19 (Fig. 15) LLcotton25 US PTA-3343 PAT (HT)20030097687 MON15985 U.S. Pat. No. PTA-2516 cry1Ac, (IR, AR, SM) ³9,133,473 cry2Ab2 MON88701 U.S. Pat. No. PTA-11754 DMO, PAT SEQ ID (HT)⁴8,735,661 NO: 20 MON88913 U.S. Pat. No. PTA-4854 cp4 epsps (HT)7,381,861 ¹ Traits: IR = Insect Resistance; HT = Herbicide Tolerance; AR= Antibiotic Resistance; SM = Screenable Marker ² Both cry1Ac cottonevent 3006-210-23 and cry1F cotton event 281-24-236 described in U.S.Pat. No. 7,179,965; seed comprising both events deposited with ATCC asPTA-6233. ³ Contains both the MON531 chimeric Cry1A and MON15985X Cry2Abinsertions. ⁴Tolerance to dicamba and glufosinate herbicides.

TABLE 4 Canola Events (transgenic loci) Patent or Patent SEQ EventApplication Trait ID NO Name Publication ATCC expression (Figure(traits) ¹ Number(s) Deposit cassette(s) Number) GT73 (HT) U.S. Pat. No.PTA-121409 cp4 epsps 8,048,632 U.S. Pat. No. 9,474,223 HCN28/T45 (HT)MON88302 U.S. Pat. No. PTA-10955 cp4 epsps SEQ ID (HT) 9,738,903 NO: 21(FIG. 16) MS8 (MS) US2003188347 PTA-730 RF3 (HT) US2003188347 PTA-730 ¹Traits: HT = Herbicide Tolerance; MS = Male Sterility

Sequences of the junction polynucleotides as well as the transgenicinsert(s) of certain transgenic loci which can be improved by themethods provided herein are set forth in Tables 1-4 (e.g., SEQ ID NO:1-34), the patent references set forth therein and incorporated hereinby reference in their entireties, and elsewhere in this disclosure. Thelocations of the 5′ and 3′ junction polynucleotides of certain maize andsoybean transgenic loci in Tables 1 and 2 are provided in Table 5. Such5′ junction polynucleotides span the junction of the 5′ plant genomicflank nucleotides and the transgenic insert nucleotides of the indicatedtransgenic events (i.e., transgenic loci) in Table 5. Such 3′ junctionpolynucleotides span the junction of the transgenic insert nucleotidesand the 3′ plant genomic flank nucleotides of the indicated transgenicevents (i.e., transgenic loci). In certain embodiments provided herein,the transgenic loci set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34) arereferred to as “original transgenic loci.” Allelic or other variantsequences corresponding to the sequences set forth in Tables 1-4 (e.g.,SEQ ID NO: 1-34), the patent references set forth therein andincorporated herein by reference in their entireties, and elsewhere inthis disclosure which may be present in certain variant transgenic plantloci can also be improved by identifying sequences in the variants thatcorrespond to the sequences of SEQ ID NO: 1-34 by performing a pairwisealignment (e.g., using CLUSTAL O 1.2.4 with default parameters) andmaking corresponding changes in the allelic or other variant sequences.Such allelic or other variant sequences include sequences having atleast 85%, 90%, 95%, 98%, or 99% sequence identity across the entirelength or at least 20, 40, 100, 500, 1,000, 2,000, 4,000, 8,000, 10,000,or 12,000 nucleotides of the sequences set forth in Tables 1-4 (e.g.,SEQ ID NO: 1-34), the patent references set forth therein andincorporated herein by reference in their entireties, and elsewhere inthis disclosure. Also provided are plants, plant parts including seeds,genomic DNA, and/or DNA obtained from plants set forth in Tables 1-4which comprise one or more modifications (e.g., via insertion of a CgRRSin a junction polynucleotide sequence) which provide for their excisionas well as transgenic loci excision sites wherein a segment comprising,consisting essentially of, or consisting of a transgenic locus orportion thereof is deleted. In certain embodiments, the transgenic lociset forth in Tables 1-4 and SEQ ID NO: 1-34 are further modified bydeletion of a segment of DNA comprising, consisting essentially of, orconsisting of a selectable marker gene and/or non-essential DNA. Alsoprovided herein are methods of detecting plants, genomic DNA, and/or DNAobtained from plants set forth in Tables 1-4 comprising a CgRRS,deletions of selectable marker genes, deletions of non-essential DNA, ora transgenic locus excision site. A first junction polynucleotide of atransgenic locus can comprise either one of the junction polynucleotidesfound at the 5′ end or the 3′ end of any one of the sequences set forthin SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof.An OgRRS can be found within non-transgenic DNA, transgenic DNA, or acombination thereof of either one of the junction polynucleotides of anyone of SEQ ID NO: 1-34, allelic variants thereof, or other variantsthereof. A second junction polynucleotide of a transgenic locus cancomprise either one of the junction polynucleotides found at the 5′ or3′ end of any one of the sequences set forth in SEQ ID NO: 1-34, allelicvariants thereof, or other variants thereof. A CgRRS can be introducedwithin transgenic DNA, non-transgenic DNA, or a combination thereof ofeither one of the junction polynucleotides of any one of SEQ ID NO:1-34, allelic variants thereof, or other variants thereof. In certainembodiments, the OgRRS is found in transgenic DNA, non-transgenic DNA,or a combination thereof in the 5′ junction polynucleotide of atransgenic locus of any one of SEQ ID NO: 1-34, allelic variantsthereof, or other variants thereof and the corresponding CgRRS isintroduced into the transgenic DNA, non-transgenic DNA, or a combinationthereof in the 3′ junction polynucleotide of the transgenic locus of SEQID NO: 1-34, allelic variants thereof, or other variants thereof. Inother embodiments, the OgRRS is found in non-transgenic DNA of the 3′junction polynucleotide of transgenic locus of any one of SEQ ID NO:1-34, allelic variants thereof, or other variants thereof and thecorresponding CgRRS is introduced into the transgenic or non-transgenicDNA of the 5′ junction polynucleotide of the transgenic locus of SEQ IDNO: 1-34, allelic variants thereof, or other variants thereof.

TABLE 5 Locations of 5′ and 3′ junction polynucleotides of certain maizeand soybean transgenic loci in Tables 1 and 2. 5′ plant 3′ plant genomicTransgene genomic flank Insert flank nucleotides Nucleotides nucleotidesSEQ of SEQ of SEQ of SEQ Event Name ID NO ID NO ID NO ID NO DAS-59122-7SEQ ID 1-2593 2594-9936  9937-11922 NO: 1 DAS-40278 SEQ ID 1-18561857-6781 6782-8557 NO: 22 DP-4114 SEQ ID 1-2422  2423-14347 14348-16752NO: 2 DP-32138 SEQ ID 1-2090  2091-11989 11990-13998 NO: 24 DP-33121 SEQID 1-398   399-24758 24759-25250 NO: 23 HCEM485 ¹ SEQ ID   1-60106011-6755 NO: 25 LY038 SEQ ID 1-1781 1782-5957 5958-6624 NO: 26 MON87403SEQ ID 1-1008 1009-4688 4689-5744 NO: 27 MON87411 SEQ ID 1-799  800-12064 12065-12248 NO: 3 MON87419 SEQ ID 1-1032 1033-8239 8240-9259NO: 28 MON87460 SEQ ID 1-1060 1061-4369 4370-5629 NO: 29 MON89034 SEQ ID1-2061  2062-11378 11379-12282 NO: 4 MIR162 SEQ ID 1-1088 1089-9390 9391-10579 NO: 5 MIR604 SEQ ID 1-801   802-9484  9485-10547 NO: 6MZHG0JG SEQ ID 1-481   482-9391 9392-9920 NO: 30 MZIR098 SEQ ID 1-10  11-8477 8478-8487 NO: 31 NK603 SEQ ID 1-260   261-7488 7489-7584 NO: 7SYN-E3272-5 SEQ ID 1-1049 1050-7059 7060-9067 NO: 8 TC1507 SEQ ID 1-669  670-10358 10359-11361 NO: 9 VCO-Ø1981- SEQ ID 1-700   701-53925393-5092 5 NO: 32 98140 SEQ ID 1-720   721-8107 8108-9425 NO: 33  5307SEQ ID 1-1348 1349-7772 7773-8865 NO: 10 DAS44406-6 SEQ ID 1-1497 1498-11771 11772-13659 NO: 11 DAS68416-4 SEQ ID 1-2730 2731-9121 9122-10212 NO: 12 DAS81419-2 SEQ ID 1-1400  1401-13896 13897-15294 NO:13 MON87701 SEQ ID 1-5757  5758-12183 12184-14416 NO: 14 MON87708 SEQ ID1-1126 1127-4129 4130-5946 NO: 15 MON89788 SEQ ID 1-1103 1104-54065407-6466 NO: 16

In certain embodiments, the CgRRS is comprised in whole or in part of anexogenous DNA molecule that is introduced into a DNA junctionpolynucleotide by genome editing. In certain embodiments, the guide RNAhybridization site of the CgRRS is operably linked to a pre-existing PAMsite in the transgenic DNA or non-transgenic DNA of the transgenic plantgenome. In other embodiments, the guide RNA hybridization site of theCgRRS is operably linked to a new PAM site that is introduced in the DNAjunction polynucleotide by genome editing. A CgRRS can be located innon-transgenic plant genomic DNA of a DNA junction polynucleotide of amodified transgenic locus, in transgenic DNA of a DNA junctionpolynucleotide of a modified transgenic locus or can span the junctionof the transgenic and non-transgenic DNA of a DNA junctionpolynucleotide of a modified transgenic locus.

Methods provided herein can be used in a variety of breeding schemes toobtain elite crop plants comprising subsets of desired modifiedtransgenic loci comprising an OgRRS and a CgRRS operably linked tojunction polynucleotide sequences and transgenic loci excision siteswhere undesired transgenic loci or portions thereof have been removed(e.g., by use of the OgRRS and a CgRRS). Such methods are useful atleast insofar as they allow for production of distinct useful donorplant lines each having unique sets of modified transgenic loci and, insome instances, targeted genetic changes that are tailored for distinctgeographies and/or product offerings. In an illustrative andnon-limiting example, a different product lines comprising transgenicloci conferring only two of three types of herbicide tolerance (e.g.,glyphosate, glufosinate, and dicamba) can be obtained from a singledonor line comprising three distinct transgenic loci conferringresistance to all three herbicides. In certain aspects, plantscomprising the subsets of undesired transgenic loci and transgenic lociexcision sites can further comprise targeted genetic changes. Such elitecrop plants can be inbred plant lines or can be hybrid plant lines. Incertain embodiments, at least two transgenic loci (e.g., transgenic lociin Tables 1-4 or modifications thereof wherein an OgRRS and a CgRRS siteis operably linked to a first and a second junction sequence andoptionally a selectable marker gene and/or non-essential DNA aredeleted) are introgressed into a desired donor line comprising elitecrop plant germplasm and then subjected to genome editing molecules torecover plants comprising one of the two introgressed transgenic loci aswell as a transgenic loci excision site introduced by excision of theother transgenic locus or portion thereof by the genome editingmolecules. In certain embodiments, the genome editing molecules can beused to remove a transgenic locus and introduce targeted genetic changesin the crop plant genome. Introgression can be achieved by backcrossingplants comprising the transgenic loci to a recurrent parent comprisingthe desired elite germplasm and selecting progeny with the transgenicloci and recurrent parent germplasm. Such backcrosses can be repeatedand/or supplemented by molecular assisted breeding techniques using SNPor other nucleic acid markers to select for recurrent parent germplasmuntil a desired recurrent parent percentage is obtained (e.g., at leastabout 95%, 96%, 97%, 98%, or 99% recurrent parent percentage). Anon-limiting, illustrative depiction of a scheme for obtaining plantswith both subsets of transgenic loci and the targeted genetic changes isshown in the FIG. 11 (bottom “Alternative” panel), where two or more ofthe transgenic loci (“Event” in FIG. 11 ) are provided in Line A andthen moved into elite crop plant germplasm by introgression. In thenon-limiting FIG. 11 illustration, introgression can be achieved bycrossing a “Line A” comprising two or more of the modified transgenicloci to the elite germplasm and then backcrossing progeny of the crosscomprising the transgenic loci to the elite germplasm as the recurrentparent) to obtain a “Universal Donor” (e.g. Line A+ in FIG. 11 )comprising two or more of the modified transgenic loci. This elitegermplasm containing the modified transgenic loci (e.g. “UniversalDonor” of FIG. 11 ) can then be subjected to genome editing moleculeswhich can excise at least one of the transgenic loci (“Event Removal” inFIG. 11 ) and introduce other targeted genetic changes (“GE” in FIG. 11) in the genomes of the elite crop plants containing one of thetransgenic loci and a transgenic locus excision site corresponding tothe removal site of one of the transgenic loci. Such selective excisionof transgenic loci or portions thereof can be effected by contacting thegenome of the plant comprising two transgenic loci with gene editingmolecules (e.g., RdDe and gRNAs, TALENS, and/or ZFN) which recognize onetransgenic loci but not another transgenic loci. Genome editingmolecules that provide for selective excision of a first modifiedtransgenic locus or portion thereof comprising an OgRRS and a CgRRSinclude a gRNA that hybridizes to the OgRRS and CgRRS of the firstmodified transgenic locus and an RdDe that recognizes the gRNA/OgRRS andgRNA/CgRRS complexes. Distinct plant lines with different subsets oftransgenic loci and desired targeted genetic changes are thus recovered(e.g., “Line B-1,” “Line B-2,” and “Line B-3” in FIG. 11 ). In certainembodiments, it is also desirable to bulk up populations of inbred elitecrop plants or their seed comprising the subset of transgenic loci and atransgenic locus excision site by selfing. Such inbred progeny of theselfed plants can be used either as is for commercial sales where thecrop can be grown a varietal, non-hybrid crop (e.g., commonly though notalways in soybean, cotton, or canola) comprising the subset of desiredtransgenic loci and one or more transgenic loci excision sites. Incertain embodiments, inbred progeny of the selfed plants can be used asa pollen donor or recipient for hybrid seed production (e.g., mostcommonly in maize but also in cotton, soybean, and canola). Such hybridseed and the progeny grown therefrom can comprise a subset of desiredtransgenic loci and a transgenic loci excision site.

Hybrid plant lines comprising elite crop plant germplasm, at least onetransgenic locus and at least one transgenic locus excision site, and incertain aspects, additional targeted genetic changes are also providedherein. Methods for production of such hybrid seed can comprise crossingelite crop plant lines where at least one of the pollen donor orrecipient comprises at least the transgenic locus and a transgenic locusexcision site and/or additional targeted genetic changes. In certainembodiments, the pollen donor and recipient will comprise germplasm ofdistinct heterotic groups and provide hybrid seed and plants exhibitingheterosis. In certain embodiments, the pollen donor and recipient caneach comprise a distinct transgenic locus which confers either adistinct trait (e.g., herbicide tolerance or insect resistance), adifferent type of trait (e.g., tolerance to distinct herbicides or todistinct insects such as coleopteran or lepidopteran insects), or adifferent mode-of-action for the same trait (e.g., resistance tocoleopteran insects by two distinct modes-of-action or resistance tolepidopteran insects by two distinct modes-of-action). In certainembodiments, the pollen recipient will be rendered male sterile orconditionally male sterile. Methods for inducing male sterility orconditional male sterility include emasculation (e.g., detasseling),cytoplasmic male sterility, chemical hybridizing agents or systems, atransgenes or transgene systems, and/or mutation(s) in one or moreendogenous plant genes. Descriptions of various male sterility systemsthat can be adapted for use with the elite crop plants provided hereinare described in Wan et al. Molecular Plant; 12, 3, (2019):321-342 aswell as in U.S. Pat. No. 8,618,358; US 20130031674; and US 2003188347.

In certain embodiments, it will be desirable to use genome editingmolecules to make modified transgenic loci by introducing a CgRRS intothe transgenic loci, to excise modified transgenic loci comprising anOgRRS and a CgRRS, and/or to make targeted genetic changes in elite cropplant or other germplasm. Techniques for effecting genome editing incrop plants (e.g., maize,) include use of morphogenic factors such asWuschel (WUS), Ovule Development Protein (ODP), and/or Babyboom (BBM)which can improve the efficiency of recovering plants with desiredgenome edits. In some embodiments, the morphogenic factor comprisesWUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, WOX9, BBM2, BMN2, BMN3, and/orODP2. In certain embodiments, compositions and methods for using WUS,BBM, and/or ODP, as well as other techniques which can be adapted foreffecting genome edits in elite crop plant and other germplasm, are setforth in US 20030082813, US 20080134353, US 20090328252, US 20100100981,US 20110165679, US 20140157453, US 20140173775, and US 20170240911,which are each incorporated by reference in their entireties. In certainembodiments, the genome edits can be effected in regenerable plant parts(e.g., plant embryos) of elite crop plants by transient provision ofgene editing molecules or polynucleotides encoding the same and do notnecessarily require incorporating a selectable marker gene into theplant genome (e.g., US 20160208271 and US 20180273960, both incorporatedherein by reference in their entireties; Svitashev et al. Nat Commun.2016; 7:13274).

In certain embodiments, edited transgenic plant genomes, transgenicplant cells, parts, or plants containing those genomes, and DNAmolecules obtained therefrom, can comprise a desired subset oftransgenic loci and/or comprise at least one transgenic locus excisionsite. In certain embodiments where a segment comprising an modifiedtransgenic locus (e.g., a transgenic locus comprising an OgRRS innon-transgenic DNA of a 1st junction sequence and a CgRRS in a 2ndjunction sequence) has been deleted by use of a gRNA and RdDe thatrecognize the OgRRS and the CgRRS, the transgenic locus excision sitecan comprise a contiguous segment of DNA comprising at least 10 basepairs of DNA that is telomere proximal to the deleted segment of thetransgenic locus and at least 10 base pairs of DNA that is centromereproximal to the deleted segment of the transgenic locus wherein thetransgenic DNA (i.e., the heterologous DNA) that has been inserted intothe crop plant genome has been deleted. In certain embodiments where asegment comprising a transgenic locus has been deleted, the transgeniclocus excision site can comprise a contiguous segment of DNA comprisingat least 10 base pairs DNA that is telomere proximal to the deletedsegment of the transgenic locus and at least 10 base pairs of DNA thatis centromere proximal DNA to the deleted segment of the transgeniclocus wherein the heterologous transgenic DNA and at least 1, 2, 5, 10,20, 50, or more base pairs of endogenous DNA located in a 5′ junctionsequence and/or in a 3′ junction sequence of the original transgeniclocus that has been deleted. In such embodiments where DNA comprisingthe transgenic locus is deleted, a transgenic locus excision site cancomprise at least 10 base pairs of DNA that is telomere proximal to thedeleted segment of the transgenic locus and at least 10 base pairs ofDNA that is centromere proximal to the deleted segment of the transgeniclocus wherein all of the transgenic DNA is absent and either all or lessthan all of the endogenous DNA flanking the transgenic DNA sequences arepresent. In certain embodiments where a segment consisting essentiallyof an original transgenic locus has been deleted, the transgenic locusexcision site can be a contiguous segment of at least 10 base pairs ofDNA that is telomere proximal to the deleted segment of the transgeniclocus and at least 10 base pairs of DNA that is centromere proximal tothe deleted segment of the transgenic locus wherein less than all of theheterologous transgenic DNA that has been inserted into the crop plantgenome is excised. In certain aforementioned embodiments where a segmentconsisting essentially of an original transgenic locus has been deleted,the transgenic locus excision site can thus contain at least 1 base pairof DNA or 1 to about 2 or 5, 8, 10, 20, or 50 base pairs of DNAcomprising the telomere proximal and/or centromere proximal heterologoustransgenic DNA that has been inserted into the crop plant genome. Incertain embodiments where a segment consisting of an original transgeniclocus has been deleted, the transgenic locus excision site can contain acontiguous segment of DNA comprising at least 10 base pairs of DNA thatis telomere proximal to the deleted segment of the transgenic locus andat least 10 base pairs of DNA that is centromere proximal to the deletedsegment of the transgenic locus wherein the heterologous transgenic DNAthat has been inserted into the crop plant genome is deleted. In certainembodiments where DNA consisting of the transgenic locus is deleted, atransgenic locus excision site can comprise at least 10 base pairs ofDNA that is telomere proximal to the deleted segment of the transgeniclocus and at least 10 base pairs of DNA that is centromere proximal tothe deleted segment of the transgenic locus wherein all of theheterologous transgenic DNA that has been inserted into the crop plantgenome is deleted and all of the endogenous DNA flanking theheterologous sequences of the transgenic locus is present. In any of theaforementioned embodiments or in other embodiments, the continuoussegment of DNA comprising the transgenic locus excision site can furthercomprise an insertion of 1 to about 2, 5, 10, 20, or more nucleotidesbetween the DNA that is telomere proximal to the deleted segment of thetransgenic locus and the DNA that is centromere proximal to the deletedsegment of the transgenic locus. Such insertions can result either fromendogenous DNA repair and/or recombination activities at the doublestranded breaks introduced at the excision site and/or from deliberateinsertion of an oligonucleotide. Plants, edited plant genomes,biological samples, and DNA molecules (e.g., including isolated orpurified DNA molecules) comprising the transgenic loci excision sitesare provided herein.

In other embodiments, a segment comprising a modified transgenic locus(e.g., a transgenic locus comprising an OgRRS in non-transgenic DNA of a1^(st) junction sequence and a CgRRS in a 2^(nd) junction sequence) canbe deleted with a gRNA and RdDe that recognize the OgRRS and the CgRRSand replaced with DNA comprising the endogenous non-transgenic plantgenomic DNA present in the genome prior to transgene insertion. Anon-limiting example of such replacements can be visualized in FIG. 17C,where the donor DNA template can comprise the endogenous non-transgenicplant genomic DNA present in the genome prior to transgene insertionalong with sufficient homology to non-transgenic DNA on each side of theexcision site to permit homology-directed repair. In certainembodiments, the endogenous non-transgenic plant genomic DNA present inthe genome prior to transgene insertion can be at least partiallyrestored. In certain embodiments, the endogenous non-transgenic plantgenomic DNA present in the genome prior to transgene insertion can beessentially restored such that no more than about 5, 10, or 20 to about50, 80, or 100 nucleotides are changed relative to the endogenous DNA atthe essentially restored excision site. In other embodiments, theendogenous non-transgenic plant genomic DNA present in the genome priorto transgene insertion can be completely restored at the excision sitesuch that it is indistinguishable from the endogenous non-transgenicplant genomic DNA present in the genome prior to transgene insertion(i.e., there are no nucleotide changes relative to the endogenous DNA atthe completely restored excision site).

In certain embodiments, modified versions of an approved transgeniclocus are provided which can comprise an OgRRS and a CgRRS which areoperably linked to a 1^(st) and a 2^(nd) junction sequences,respectively, and can optionally further comprise deletions ofselectable marker genes. In their unmodified form (in certainembodiments, the “unmodified form” is the “original form,” “originaltransgenic locus,” etc.) many approved transgenic loci comprises atleast one selectable marker gene. In a modified version, at least oneselectable marker has been deleted with genome editing molecules asdescribed elsewhere herein from the unmodified approved transgeniclocus. In certain embodiments, the deletion of the selectable markergene does not affect any other functionality of the approved transgeniclocus. In certain embodiments, the selectable marker gene that isdeleted confers resistance to an antibiotic, tolerance to an herbicide,or an ability to grow on a specific carbon source, for example, mannose.In certain embodiments, the selectable marker gene comprises a DNAencoding a phosphinothricin acetyl transferase (PAT), a glyphosatetolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), aglyphosate oxidase (GOX), neomycin phosphotransferase (npt), ahygromycin phosphotransferase (hyg), an aminoglycoside adenyltransferase, or a phosphomannose isomerase (pmi). In certainembodiments, the modified locus does not contain a site-specificrecombination system DNA recognition site, for example, in certainembodiments, the modified locus does not contain a lox or FRT site. Incertain embodiments, the selectable marker gene to be deleted is flankedby operably linked protospacer adjacent motif (PAM) sites in theunmodified form of the approved transgenic locus. Thus, in certainembodiments of the modified locus, PAM sites flank the excision site ofthe deleted selectable marker gene. In certain embodiments, the PAMsites are recognized by an RNA dependent DNA endonuclease (RdDe); forexample, a class 2 type II or class 2 type V RdDe. In certainembodiments, the deleted selectable marker gene is replaced in themodified approved transgenic locus by an introduced DNA sequence asdiscussed in further detail elsewhere herein. For example, in certainembodiments, the introduced DNA sequence comprises a trait expressioncassette such as a trait expression cassette of another transgeniclocus. In addition to the deletion of a selectable marker gene, incertain embodiments at least one copy of a repetitive sequence has alsobeen deleted with genome editing molecules from an unmodified approvedtransgenic locus. In certain embodiments, deletion of the repetitivesequence enhances the functionality of the modified approved transgeniclocus. In certain embodiments, the approved transgenic locus which ismodified is: (i) a Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411,MON87427, MON88017, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278,DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87403,MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, and/or TC1507transgenic locus in a transgenic maize plant genome; (ii) an A5547-127,DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708,MON89788, MST-FGØ72-3, and/or SYHT0H2 transgenic locus in a transgenicsoybean plant genome; (iii) a DAS-21023-5, DAS-24236-5, COT102,LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locus in atransgenic cotton plant genome; or (iv) a GT73, HCN28, MON88302, and/orMS8 transgenic locus in a transgenic canola plant genome. Also providedherein are plants comprising any of aforementioned modified transgenicloci.

In certain embodiments, edited transgenic plant genomes and transgenicplant cells, plant parts, or plants containing those edited genomes,comprising a modification of an original transgenic locus, where themodification comprises an OgRRS and a CgRRS which are operably linked toa 1^(st) and a 2^(nd) junction sequence, respectively, and optionallyfurther comprise a deletion of a segment of the original transgeniclocus. In certain embodiments, the modification comprises two or moreseparate deletions and/or there is a modification in two or moreoriginal transgenic plant loci. In certain embodiments, the deletedsegment comprises, consists essentially of, or consists of a segment ofnon-essential DNA in the transgenic locus. Illustrative examples ofnon-essential DNA include but are not limited to synthetic cloning sitesequences, duplications of transgene sequences; fragments of transgenesequences, and Agrobacterium right and/or left border sequences. Incertain embodiments, the non-essential DNA is a duplication and/orfragment of a promoter sequence and/or is not the promoter sequenceoperably linked in the cassette to drive expression of a transgene. Incertain embodiments, excision of the non-essential DNA improves acharacteristic, functionality, and/or expression of a transgene of thetransgenic locus or otherwise confers a recognized improvement in atransgenic plant comprising the edited transgenic plant genome. Incertain embodiments, the non-essential DNA does not comprise DNAencoding a selectable marker gene. In certain embodiments of an editedtransgenic plant genome, the modification comprises a deletion of thenon-essential DNA and a deletion of a selectable marker gene. Themodification producing the edited transgenic plant genome could occur byexcising both the non-essential DNA and the selectable marker gene atthe same time, e.g., in the same modification step, or the modificationcould occur step-wise. For example, an edited transgenic plant genome inwhich a selectable marker gene has previously been removed from thetransgenic locus can comprise an original transgenic locus from which anon-essential DNA is further excised and vice versa. In certainembodiments, the modification comprising deletion of the non-essentialDNA and deletion of the selectable marker gene comprises excising asingle segment of the original transgenic locus that comprises both thenon-essential DNA and the selectable marker gene. Such modificationwould result in one excision site in the edited transgenic genomecorresponding to the deletion of both the non-essential DNA and theselectable marker gene. In certain embodiments, the modificationcomprising deletion of the non-essential DNA and deletion of theselectable marker gene comprises excising two or more segments of theoriginal transgenic locus to achieve deletion of both the non-essentialDNA and the selectable marker gene. Such modification would result in atleast two excision sites in the edited transgenic genome correspondingto the deletion of both the non-essential DNA and the selectable markergene. In certain embodiments of an edited transgenic plant genome, priorto excision, the segment to be deleted is flanked by operably linkedprotospacer adjacent motif (PAM) sites in the original or unmodifiedtransgenic locus and/or the segment to be deleted encompasses anoperably linked PAM site in the original or unmodified transgenic locus.In certain embodiments, following excision of the segment, the resultingedited transgenic plant genome comprises PAM sites flanking the deletionsite in the modified transgenic locus. In certain embodiments of anedited transgenic plant genome, the modification comprises amodification of a Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411,MON87427, MON88017, MON89034, MIR162, MIR604, NK603, SYN-E3272-5, 5307,DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403,MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140,and/or TC1507 original transgenic locus in a transgenic corn plantgenome. In certain embodiments of an edited transgenic plant genome, themodification comprises a modification of an A5547-127, DAS44406-6,DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,MST-FGØ72-3, and/or SYHT0H2 original transgenic locus in a transgenicsoybean plant genome. In certain embodiments of an edited transgenicplant genome, the modification comprises a modification of aDAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/orMON88913 original transgenic locus in a transgenic cotton plant genome.In certain embodiments of an edited transgenic plant genome, themodification comprises a modification of an GT73, HCN28, MON88302,and/or MS8 original transgenic locus in a transgenic canola plantgenome.

Nucleic acid markers adapted for detecting the transgenic loci excisionsites as well as methods for detecting the presence of DNA moleculescomprising the transgenic loci excision sites are also provided herein.Methods and reagents (e.g., nucleic acid markers including nucleic acidprobes and/or primers) for detecting plants, edited plant genomes, andbiological samples containing DNA molecules comprising the transgenicloci excision sites and/or non-essential DNA deletions are also providedherein. Detection of the DNA molecules can be achieved by anycombination of nucleic acid amplification (e.g., PCR amplification),hybridization, sequencing, and/or mass-spectrometry based techniques.Methods set forth for detecting junction nucleic acids in unmodifiedtransgenic loci set forth in US 20190136331 and U.S. Pat. No. 9,738,904,both incorporated herein by reference in their entireties, can beadapted for use in detection of the nucleic acids provided herein. Incertain embodiments, such detection is achieved by amplification and/orhybridization-based detection methods using a method (e.g., selectiveamplification primers) and/or probe (e.g., capable of selectivehybridization or generation of a specific primer extension product)which specifically recognizes the target DNA molecule (e.g., transgeniclocus excision site) but does not recognize DNA from an unmodifiedtransgenic locus. In certain embodiments, the hybridization probes cancomprise detectable labels (e.g., fluorescent, radioactive, epitope, andchemiluminescent labels). In certain embodiments, a single nucleotidepolymorphism detection assay can be adapted for detection of the targetDNA molecule (e.g., transgenic locus excision site).

In certain embodiments, improvements in a transgenic plant locus areobtained by introducing a new cognate guide RNA recognition site (CgRRS)which is operably linked to a DNA junction polynucleotide of thetransgenic locus in the transgenic plant genome. Such CgRRS sites can berecognized by RdDe and a single suitable guide RNA directed to the CgRRSand the originator gRNA Recognition Site (OgRRS) to provide for cleavagewithin the junction polynucleotides which flank a modified transgeniclocus. In certain embodiments, the CgRRS/gRNA and OgRRS/gRNAhybridization complexes are recognized by the same class of RdDe (e.g.,Class 2 type II or Class 2 type V) or by the same RdDe (e.g., both theCgRRS/gRNA and OgRRS/gRNA hybridization complexes recognized by the sameCas9 or Cas 12 RdDe). Such CgRRS and OgRRS can be recognized by RdDe andsuitable guide RNAs containing crRNA sufficiently complementary to theguide RNA hybridization site DNA sequences adjacent to the PAM site ofthe CgRRS and the OgRRS to provide for cleavage within or near the twojunction polynucleotides. Suitable guide RNAs can be in the form of asingle gRNA comprising a crRNA or in the form of a crRNA/tracrRNAcomplex. In the case of the OgRRS site, the PAM and guide RNAhybridization site are endogenous DNA polynucleotide molecules found inthe plant genome. In certain embodiments where the CgRRS is introducedinto the plant genome by genome editing, gRNA hybridization sitepolynucleotides introduced at the CgRRS are at least 17 or 18nucleotides in length and are complementary to the crRNA of a guide RNA.In certain embodiments, the gRNA hybridization site sequence of theOgRRS and/or the CgRRS is about 17 or 18 to about 24 nucleotides inlength. The gRNA hybridization site sequence of the OgRRS and the gRNAhybridization site of the CgRRS can be of different lengths or comprisedifferent sequences so long as there is sufficient complementarity topermit hybridization by a single gRNA and recognition by a RdDe thatrecognizes and cleaves DNA at the gRNA/OgRRS and gRNA/CgRRS complex. Incertain embodiments, the guide RNA hybridization site of the CgRRScomprise about a 17 or 18 to about 24 nucleotide sequence which isidentical to the guide RNA hybridization site of the OgRRS. In otherembodiments, the guide RNA hybridization site of the CgRRS compriseabout a 17 or 18 to about 24 nucleotide sequence which has one, two,three, four, or five nucleotide insertions, deletions or substitutionswhen compared to the guide RNA hybridization site of the OgRRS. CertainCgRRS comprising a gRNA hybridization site containing has one, two,three, four, or five nucleotide insertions, deletions or substitutionswhen compared to the guide RNA hybridization site of the OgRRS canundergo hybridization with a gRNA which is complementary to the OgRRSgRNA hybridization site and be cleaved by certain RdDe. Examples ofmismatches between gRNAs and guide RNA hybridization sites which allowfor RdDe recognition and cleavage include mismatches resulting from bothnucleotide insertions and deletions in the DNA which is hybridized tothe gRNA (e.g., Lin et al., doi: 10.1093/nar/gku402). In certainembodiments, an operably linked PAM site is co-introduced with the gRNAhybridization site polynucleotide at the CgRRS. In certain embodiments,the gRNA hybridization site polynucleotides are introduced at a positionadjacent to a resident endogenous PAM sequence in the junctionpolynucleotide sequence to form a CgRRS where the gRNA hybridizationsite polynucleotides are operably linked to the endogenous PAM site. Incertain embodiments, non-limiting features of the OgRRS, CgRRS, and/orthe gRNA hybridization site polynucleotides thereof include: (i) absenceof significant homology or sequence identity (e.g., less than 50%sequence identity across the entire length of the OgRRS, CgRRS, and/orthe gRNA hybridization site sequence) to any other endogenous ortransgenic sequences present in the transgenic plant genome or in othertransgenic genomes of the particular crop plant being transformed andedited (e.g., corn, soybean, cotton, canola, rice, wheat, and the like);(ii) absence of significant homology or sequence identity (e.g., lessthan 50% sequence identity across the entire length of the sequence) ofa sequence of a first OgRRS and a first CgRRS to a second OgRRS and asecond CgRRS which are operably linked to junction polynucleotides of adistinct transgenic locus; (iii) the presence of some sequence identity(e.g., about 25%, 40%, or 50% to about 60%, 70%, or 80%) between theOgRRS sequence and endogenous sequences present at the site where theCgRRS sequence is introduced; and/or (iv) optimization of the gRNAhybridization site polynucleotides for recognition by the RdDe and guideRNA when used in conjunction with a particular PAM sequence. In certainembodiments, the first and second OgRRS as well as the first and secondCgRRS are recognized by the same class of RdDe (e.g., Class 2 type II orClass 2 type V) or by the same RdDe (e.g., Cas9 or Cas 12 RdDe). Incertain embodiments, the first OgRRS site in a first junctionpolynucleotide and the CgRRS introduced in the second junctionpolynucleotide to permit excision of a first transgenic locus or portionthereof by a first single guide RNA and a single RdDe. Such nucleotideinsertions or genome edits used to introduce CgRRS in a transgenic plantgenome can be effected in the plant genome by using gene editingmolecules (e.g., RdDe and guide RNAs, RNA dependent nickases and guideRNAs, Zinc Finger nucleases or nickases, or TALE nucleases or nickases)which introduce blunt double stranded breaks or staggered doublestranded breaks in the DNA junction polynucleotides. In the case of DNAinsertions, the genome editing molecules can also in certain embodimentsfurther comprise a donor DNA template or other DNA template whichcomprises the heterologous nucleotides for insertion to form the CgRRS.Guide RNAs can be directed to the junction polynucleotides by using apre-existing PAM site located within or adjacent to a junctionpolynucleotide of the transgenic locus. Non-limiting examples of suchpre-existing PAM sites present in junction polynucleotides, which can beused either in conjunction with an inserted heterologous sequence toform a CgRRS or which can be used to create a double stranded break toinsert or create a CgRRS, include PAM sites recognized by a Cas9 orCas12a enzyme. Non-limiting examples where CgRRS are created in a DNAsequence are illustrated in the Examples.

Transgenic loci or portions thereof comprising OgRRS and CgRRS in afirst and a second junction polynucleotides can be excised from thegenomes of transgenic plants by contacting the transgenic loci with RdDeor RNA directed nickases, and a suitable guide RNA directed to the OgRRSand CgRRS. A non-limiting example where a modified transgenic locus isexcised from a plant genome by use of a gRNA and an RdDe that recognizesan OgRRS/gRNA and a CgRRS/gRNA complex and introduces dsDNA breaks inboth junction polynucleotides nd repaired by NHEJ is depicted in FIG.17B. In the depicted example set forth in FIG. 17B, the OgRRS site andthe CgRRS site are absent from the plant chromosome comprising thetransgene excision site that results from the process. In otherembodiments provided herein where a modified transgenic locus is excisedfrom a plant genome by use of a gRNA and an RdDe that recognizes anOgRRS/gRNA and a CgRRS/gRNA complex and repaired by NHEJ ormicrohomology-mediated end joining (MMEJ), the OgRRS and/or othernon-transgenic sequences that were originally present prior to transgeneinsertion are partially, essentially, or completely restored.

In certain embodiments, edited transgenic plant genomes provided hereincan lack one or more selectable and/or scoreable markers found in anoriginal event (transgenic locus). Original transgenic loci (events),including those set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34), thepatent references set forth therein and incorporated herein by referencein their entireties, and depicted in the drawings, can containselectable transgenes markers conferring herbicide tolerance, antibioticresistance, or an ability to grow on a carbon source. Selectable markertransgenes which can confer herbicide tolerance include genes encoding aphosphinothricin acetyl transferase (PAT), a glyphosate tolerant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), and a glyphosateoxidase (GOX). Selectable marker transgenes which can confer antibioticresistance include genes encoding a neomycin phosphotransferase (npt), ahygromycin phosphotransferase, and an aminoglycoside adenyl transferase.Transgenes encoding a phosphomannose isomerase (pmi) can confer theability to grow on mannose. Original transgenic loci (events), includingthose set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34) and the patentreferences set forth therein which are incorporated herein by referencein their entireties, can contain scoreable transgenic markers which canbe detected by enzymatic, histochemical, or other assays. Scoreablemarkers can include genes encoding beta-glucuronidase (uid) orfluorescent proteins (e.g., a GFP, RFP, or YFP). Such selectable orscoreable marker transgenes can be excised from an original transgeniclocus by contacting the transgenic locus with one or more gene editingmolecules which introduce double stranded breaks in the transgenic locusat the 5′ and 3′ end of the expression cassette comprising theselectable marker transgene (e.g., an RdDe and guide RNAs directed toPAM sites located at the 5′ and 3′ end of the expression cassettecomprising the selectable marker transgenes) and selecting for plantcells, plant parts, or plants wherein the selectable or scoreable markerhas been excised. In certain embodiments, the selectable or scoreablemarker transgene can be inactivated. Inactivation can be achieved bymodifications including insertion, deletion, and/or substitution of oneor more nucleotides in a promoter element, 5′ or 3′ untranslated region(UTRs), intron, coding region, and/or 3′ terminator and/orpolyadenylation site of the selectable marker transgene. Suchmodifications can inactivate the selectable or scoreable markertransgene by eliminating or reducing promoter activity, introducing amissense mutation, and/or introducing a pre-mature stop codon. Incertain embodiments, the selectable and/or scoreable marker transgenecan be replaced by an introduced transgene. In certain embodiments, anoriginal transgenic locus that was contacted with gene editing moleculeswhich introduce double stranded breaks in the transgenic locus at the 5′and 3′ end of the expression cassette comprising the selectable markerand/or scoreable transgene can also be contacted with a suitable donorDNA template comprising an expression cassette flanked by DNA homologousto remaining DNA in the transgenic locus located 5′ and 3′ to theselectable marker excision site. In certain embodiments, a coding regionof the selectable and/or scoreable marker transgene can be replaced withanother coding region such that the replacement coding region isoperably linked to the promoter and 3′ terminator or polyadenylationsite of the selectable and/or scoreable marker transgene.

In certain embodiments, edited transgenic plant genomes provided hereincan comprise additional new introduced transgenes (e.g., expressioncassettes) inserted into the transgenic locus of a given event.Introduced transgenes inserted at the transgenic locus of an eventsubsequent to the event's original isolation can be obtained by inducinga double stranded break at a site within an original transgenic locus(e.g., with genome editing molecules including an RdDe and suitableguide RNA(s); a suitable engineered zinc-finger nuclease; a TALENprotein and the like) and providing an exogenous transgene in a donorDNA template which can be integrated at the site of the double strandedbreak (e.g. by homology-directed repair (HDR) or by non-homologousend-joining (NHEJ)). In certain embodiments, an OgRRS and a CgRRSlocated in a 1^(st) junction polynucleotide and a 2^(nd) junctionpolynucleotide, respectively, can be used to delete the transgenic locusand replace it with one or more new expression cassettes. In certainembodiments, such deletions and replacements are effected by introducingdsDNA breaks in both junction polynucleotides and providing the newexpression cassettes on a donor DNA template (e.g., in FIG. 17C, thedonor DNA template can comprise an expression cassette flanked by DNAhomologous to non-transgenic DNA located telomere proximal andcentromere proximal to the excision site). Suitable expression cassettesfor insertion include DNA molecules comprising promoters which areoperably linked to DNA encoding proteins and/or RNA molecules whichconfer useful traits which are in turn operably linked topolyadenylation sites or terminator elements. In certain embodiments,such expression cassettes can also comprise 5′ UTRs, 3′ UTRs, and/orintrons. Useful traits include biotic stress tolerance (e.g., insectresistance, nematode resistance, or disease resistance), abiotic stresstolerance (e.g., heat, cold, drought, and/or salt tolerance), herbicidetolerance, and quality traits (e.g., improved fatty acid compositions,protein content, starch content, and the like). Suitable expressioncassettes for insertion include expression cassettes contained in any ofthe events (transgenic loci) listed in Tables 1-4 (e.g., SEQ ID NO:1-34), the patent references set forth therein and incorporated hereinby reference in their entireties or set forth in the drawings whichconfer insect resistance, herbicide tolerance, biofuel use, or malesterility traits.

In certain embodiments, plants provided herein, including plants withone or more transgenic loci, modified transgenic loci, and/or comprisingtransgenic loci excision sites can further comprise one or more targetedgenetic changes introduced by one or more of gene editing molecules orsystems. Also provided are methods where the targeted genetic changesand one or more transgenic loci excision sites are removed from plantseither in series or in parallel (e.g., as set forth in the non-limitingillustration in FIG. 11 , bottom “Alternative” panel, where “GE” canrepresent targeted genetic changes induced by gene editing molecules and“Event Removal” represents excision of one or more transgenic loci withgene editing molecules). Such targeted genetic changes include thoseconferring traits such as improved yield, improved food and/or feedcharacteristics (e.g., improved oil, starch, protein, or amino acidquality or quantity), improved nitrogen use efficiency, improved biofueluse characteristics (e.g., improved ethanol production), malesterility/conditional male sterility systems (e.g., by targetingendogenous MS26, MS45 and MSCA1 genes), herbicide tolerance (e.g., bytargeting endogenous ALS, EPSPS, HPPD, or other herbicide target genes),delayed flowering, non-flowering, increased biotic stress resistance(e.g., resistance to insect, nematode, bacterial, or fungal damage),increased abiotic stress resistance (e.g., resistance to drought, cold,heat, metal, or salt), enhanced lodging resistance, enhanced growthrate, enhanced biomass, enhanced tillering, enhanced branching, delayedflowering time, delayed senescence, increased flower number, improvedarchitecture for high density planting, improved photosynthesis,increased root mass, increased cell number, improved seedling vigor,improved seedling size, increased rate of cell division, improvedmetabolic efficiency, and increased meristem size in comparison to acontrol plant lacking the targeted genetic change. Types of targetedgenetic changes that can be introduced include insertions, deletions,and substitutions of one or more nucleotides in the crop plant genome.Sites in endogenous plant genes for the targeted genetic changes includepromoter, coding, and non-coding regions (e.g., 5′ UTRs, introns, splicedonor and acceptor sites and 3′ UTRs). In certain embodiments, thetargeted genetic change comprises an insertion of a regulatory or otherDNA sequence in an endogenous plant gene. Non-limiting examples ofregulatory sequences which can be inserted into endogenous plant geneswith gene editing molecules to effect targeted genetic changes whichconfer useful phenotypes include those set forth in US PatentApplication Publication 20190352655, which is incorporated herein byexample, such as: (a) auxin response element (AuxRE) sequence; (b) atleast one D1-4 sequence (Ulmasov et al. (1997) Plant Cell, 9:1963-1971),(c) at least one DR5 sequence (Ulmasov et al. (1997) Plant Cell,9:1963-1971); (d) at least one m5-DR5 sequence (Ulmasov et al. (1997)Plant Cell, 9:1963-1971); (e) at least one P3 sequence; (f) a small RNArecognition site sequence bound by a corresponding small RNA (e.g., ansiRNA, a microRNA (miRNA), a trans-acting siRNA as described in U.S.Pat. No. 8,030,473, or a phased sRNA as described in U.S. Pat. No.8,404,928; both of these cited patents are incorporated by referenceherein); (g) a microRNA (miRNA) recognition site sequence; (h) thesequence recognizable by a specific binding agent includes a microRNA(miRNA) recognition sequence for an engineered miRNA wherein thespecific binding agent is the corresponding engineered mature miRNA; (i)a transposon recognition sequence; (j) a sequence recognized by anethylene-responsive element binding-factor-associated amphiphilicrepression (EAR) motif; (k) a splice site sequence (e.g., a donor site,a branching site, or an acceptor site; see, for example, the splicesites and splicing signals set forth in the internet sitelemur[dot]amu[dot]edu[dot]pl/share/ERISdb/home.html); (1) a recombinaserecognition site sequence that is recognized by a site-specificrecombinase; (m) a sequence encoding an RNA or amino acid aptamer or anRNA riboswitch, the specific binding agent is the corresponding ligand,and the change in expression is upregulation or downregulation; (n) ahormone responsive element recognized by a nuclear receptor or ahormone-binding domain thereof; (o) a transcription factor bindingsequence; and (p) a polycomb response element (see Xiao et al. (2017)Nature Genetics, 49:1546-1552, doi: 10.1038/ng.3937). Non limitingexamples of target maize genes that can be subjected to targeted geneedits to confer useful traits include: (a) ZmIPK1 (herbicide tolerantand phytate reduced maize; Shukla et al., Nature. 2009; 459:437-41); (b)ZmGL2 (reduced epicuticular wax in leaves; Char et al. Plant BiotechnolJ. 2015; 13:1002); (c) ZmMTL (induction of haploid plants; Kelliher etal. Nature. 2017; 542:105); (d) Wx1 (high amylopectin content; US20190032070; incorporated herein by reference in its entirety); (e) TMSS(thermosensitive male sterile; Li et al. J Genet Genomics. 2017;44:465-8); (f) ALS (herbicide tolerance; Svitashev et al.; PlantPhysiol. 2015; 169:931-45); and (g) ARGOS8 (drought stress tolerance;Shi et al., Plant Biotechnol J. 2017; 15:207-16). Non-limiting examplesof target soybean genes that can be subjected to targeted gene edits toconfer useful traits include: (a) FAD2-1A, FAD2-1B (increased oleic acidcontent; Haun et al.; Plant Biotechnol J. 2014; 12:934-40); (b) FAD2-1A,FAD2-1B, FAD3A (increased oleic acid and decreased linolenic content;Demorest et al., BMC Plant Biol. 2016; 16:225); and (c) ALS (herbicidetolerance; Svitashev et al.; Plant Physiol. 2015; 169:931-45). Anon-limiting examples of target Brassica genes that can be subjected totargeted gene edits to confer useful traits include: (a) the FRIGIDAgene to confer early flowering (Sun Z, et al. J Integr Plant Biol. 2013;55:1092-103); and (b) ALS (herbicide tolerance; US 20160138040,incorporated herein by reference in its entirety). Non-limiting examplesof target genes in crop plants including corn and soybean which can besubjected to targeted genetic changes which confer useful phenotypesinclude those set forth in US Patent Application Nos. 20190352655,20200199609, 20200157554, and 20200231982, which are each incorporatedherein in their entireties; and Zhang et al. (Genome Biol. 2018; 19:210).

Gene editing molecules of use in methods provided herein includemolecules capable of introducing a double-strand break (“DSB”) orsingle-strand break (“SSB”) in double-stranded DNA, such as in genomicDNA or in a target gene located within the genomic DNA as well asaccompanying guide RNA or donor DNA template polynucleotides. Examplesof such gene editing molecules include: (a) a nuclease comprising anRNA-guided nuclease, an RNA-guided DNA endonuclease or RNA directed DNAendonuclease (RdDe), a class 1 CRISPR type nuclease system, a type IICas nuclease, a Cas9, a nCas9 nickase, a type V Cas nuclease, a Cas12anuclease, a nCas12a nickase, a Cas12d (CasY), a Cas12e (CasX), a Cas12b(C2c1), a Cas12c (C2c3), a Cas12i, a Cas12j, a Cas14, an engineerednuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN) ornickase, a transcription activator-like effector nuclease (TAL-effectornuclease or TALEN) or nickase (TALE-nickase), an Argonaute, and ameganuclease or engineered meganuclease; (b) a polynucleotide encodingone or more nucleases capable of effectuating site-specific alteration(including introduction of a DSB or SSB) of a target nucleotidesequence; (c) a guide RNA (gRNA) for an RNA-guided nuclease, or a DNAencoding a gRNA for an RNA-guided nuclease; (d) donor DNA templatepolynucleotides; and (e) other DNA templates (dsDNA, ssDNA, orcombinations thereof) suitable for insertion at a break in genomic DNA(e.g., by non-homologous end joining (NHEJ) or microhomology-mediatedend joining (MMEJ).

CRISPR-type genome editing can be adapted for use in the plant cells andmethods provided herein in several ways. CRISPR elements, e.g., geneediting molecules comprising CRISPR endonucleases and CRISPR guide RNAsincluding single guide RNAs or guide RNAs in combination with tracrRNAsor scoutRNA, or polynucleotides encoding the same, are useful ineffectuating genome editing without remnants of the CRISPR elements orselective genetic markers occurring in progeny. In certain embodiments,the CRISPR elements are provided directly to the eukaryotic cell (e.g.,plant cells), systems, methods, and compositions as isolated molecules,as isolated or semi-purified products of a cell free synthetic process(e.g., in vitro translation), or as isolated or semi-purified productsof in a cell-based synthetic process (e.g., such as in a bacterial orother cell lysate). In certain embodiments, genome-inserted CRISPRelements are useful in plant lines adapted for use in the methodsprovide herein. In certain embodiments, plants or plant cells used inthe systems, methods, and compositions provided herein can comprise atransgene that expresses a CRISPR endonuclease (e.g., a Cas9, aCpf1-type or other CRISPR endonuclease). In certain embodiments, one ormore CRISPR endonucleases with unique PAM recognition sites can be used.Guide RNAs (sgRNAs or crRNAs and a tracrRNA) to form an RNA-guidedendonuclease/guide RNA complex which can specifically bind sequences inthe gDNA target site that are adjacent to a protospacer adjacent motif(PAM) sequence. The type of RNA-guided endonuclease typically informsthe location of suitable PAM sites and design of crRNAs or sgRNAs.G-rich PAM sites, e.g., 5′-NGG are typically targeted for design ofcrRNAs or sgRNAs used with Cas9 proteins. Examples of PAM sequencesinclude 5′-NGG (Streptococcus pyogenes), 5′-NNAGAA (Streptococcusthermophilus CRISPR1), 5′-NGGNG (Streptococcus thermophilus CRISPR3),5′-NNGRRT or 5′-NNGRR (Staphylococcus aureus Cas9, SaCas9), and5′-NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5′-TTN or5′-TTTV, where “V” is A, C, or G) are typically targeted for design ofcrRNAs or sgRNAs used with Cas12a proteins. In some instances, Cas12acan also recognize a 5′-CTA PAM motif. Other examples of potentialCas12a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN,ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN,GCCN, and CCGN (wherein N is defined as any nucleotide). Cpf1endonuclease and corresponding guide RNAs and PAM sites are disclosed inUS Patent Application Publication 2016/0208243 A1, which is incorporatedherein by reference for its disclosure of DNA encoding Cpf1endonucleases and guide RNAs and PAM sites. Introduction of one or moreof a wide variety of CRISPR guide RNAs that interact with CRISPRendonucleases integrated into a plant genome or otherwise provided to aplant is useful for genetic editing for providing desired phenotypes ortraits, for trait screening, or for gene editing mediated traitintrogression (e.g., for introducing a trait into a new genotype withoutbackcrossing to a recurrent parent or with limited backcrossing to arecurrent parent). Multiple endonucleases can be provided in expressioncassettes with the appropriate promoters to allow multiple genome siteediting.

CRISPR technology for editing the genes of eukaryotes is disclosed in USPatent Application Publications 2016/0138008A1 and US2015/0344912A1, andin U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233,8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814,8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAsand PAM sites are disclosed in US Patent Application Publication2016/0208243 A1. Other CRISPR nucleases useful for editing genomesinclude Cas12b and Cas12c (see Shmakov et al. (2015) Mol. Cell,60:385-397; Harrington et al. (2020) Molecular Celldoi:10.1016/j.molcel.2020.06.022) and CasX and CasY (see Burstein et al.(2016) Nature, doi:10.1038/nature21059; Harrington et al. (2020)Molecular Cell doi:10.1016/j.molcel.2020.06.022), or Cas12j (Pausch etal, (2020) Science 10.1126/science.abb1400). Plant RNA promoters forexpressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9endonuclease are disclosed in International Patent ApplicationPCT/US2015/018104 (published as WO 2015/131101 and claiming priority toU.S. Provisional Patent Application 61/945,700). Methods of using CRISPRtechnology for genome editing in plants are disclosed in US PatentApplication Publications US 2015/0082478A1 and US 2015/0059010A1 and inInternational Patent Application PCT/US2015/038767 A1 (published as WO2016/007347 and claiming priority to U.S. Provisional Patent Application62/023,246). All of the patent publications referenced in this paragraphare incorporated herein by reference in their entirety. In certainembodiments, an RNA-guided endonuclease that leaves a blunt endfollowing cleavage of the target site is used. Blunt-end cuttingRNA-guided endonucleases include Cas9, Cas12c, and Cas 12h (Yan et al.,2019). In certain embodiments, an RNA-guided endonuclease that leaves astaggered single stranded DNA overhanging end following cleavage of thetarget site following cleavage of the target site is used. Staggered-endcutting RNA-guided endonucleases include Cas12a, Cas12b, and Cas12e.

The methods can also use sequence-specific endonucleases orsequence-specific endonucleases and guide RNAs that cleave a single DNAstrand in a dsDNA target site. Such cleavage of a single DNA strand in adsDNA target site is also referred to herein and elsewhere as “nicking”and can be affected by various “nickases” or systems that provide fornicking. Nickases that can be used include nCas9 (Cas9 comprising a D10Aamino acid substitution), nCas12a (e.g., Cas12a comprising an R1226Aamino acid substitution; Yamano et al., 2016), Cas12i (Yan et al. 2019),a zinc finger nickase e.g., as disclosed in Kim et al., 2012), a TALEnickase (e.g., as disclosed in Wu et al., 2014), or a combinationthereof. In certain embodiments, systems that provide for nicking cancomprise a Cas nuclease (e.g., Cas9 and/or Cas12a) and guide RNAmolecules that have at least one base mismatch to DNA sequences in thetarget editing site (Fu et al., 2019). In certain embodiments, genomemodifications can be introduced into the target editing site by creatingsingle stranded breaks (i.e., “nicks”) in genomic locations separated byno more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 basepairs of DNA. In certain illustrative and non-limiting embodiments, twonickases (i.e., a CAS nuclease which introduces a single stranded DNAbreak including nCas9, nCas12a, Cas12i, Cas 12j, zinc finger nickases,TALE nickases, combinations thereof, and the like) or nickase systemscan directed to make cuts to nearby sites separated by no more thanabout 10, 20, 30, 40, 50, 60, 80 or 100 base pairs of DNA. In instanceswhere an RNA guided nickase and an RNA guide are used, the RNA guidesare adjacent to PAM sequences that are sufficiently close (i.e.,separated by no more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or200 base pairs of DNA). For the purposes of gene editing, CRISPR arrayscan be designed to contain one or multiple guide RNA sequencescorresponding to a desired target DNA sequence; see, for example, Conget al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols,8:2281-2308. At least 16 or 17 nucleotides of gRNA sequence are requiredby Cas9 for DNA cleavage to occur; for Cpf1 at least 16 nucleotides ofgRNA sequence are needed to achieve detectable DNA cleavage and at least18 nucleotides of gRNA sequence were reported necessary for efficientDNA cleavage in vitro; see Zetsche et al. (2015) Cell, 163:759-771. Inpractice, guide RNA sequences are generally designed to have a length of17-24 nucleotides (frequently 19, 20, or 21 nucleotides) and exactcomplementarity (i.e., perfect base-pairing) to the targeted gene ornucleic acid sequence; guide RNAs having less than 100% complementarityto the target sequence can be used (e.g., a gRNA with a length of 20nucleotides and 1-4 mismatches to the target sequence) but can increasethe potential for off-target effects. The design of effective guide RNAsfor use in plant genome editing is disclosed in US Patent ApplicationPublication 2015/0082478 A1, the entire specification of which isincorporated herein by reference. More recently, efficient gene editinghas been achieved using a chimeric “single guide RNA” (“sgRNA”), anengineered (synthetic) single RNA molecule that mimics a naturallyoccurring crRNA-tracrRNA complex and contains both a tracrRNA (forbinding the nuclease) and at least one crRNA (to guide the nuclease tothe sequence targeted for editing); see, for example, Cong et al. (2013)Science, 339:819-823; Xing et al. (2014) BMC Plant Biol., 14:327-340.Chemically modified sgRNAs have been demonstrated to be effective ingenome editing; see, for example, Hendel et al. (2015) NatureBiotechnol., 985-991. The design of effective gRNAs for use in plantgenome editing is disclosed in US Patent Application Publication2015/0082478 A1, the entire specification of which is incorporatedherein by reference.

Genomic DNA may also be modified via base editing. Both adenine baseeditors (ABE) which convert A/T base pairs to G/C base pairs in genomicDNA as well as cytosine base pair editors (CBE) which effect C to Tsubstitutions can be used in certain embodiments of the methods providedherein. In certain embodiments, useful ABE and CBE can comprise genomesite specific DNA binding elements (e.g., RNA-dependent DNA bindingproteins including catalytically inactive Cas9 and Cas12 proteins orCas9 and Cas12 nickases) operably linked to adenine or cytidinedeaminases and used with guide RNAs which position the protein near thenucleotide targeted for substitution. Suitable ABE and CBE disclosed inthe literature (Kim, Nat Plants, 2018 March; 4(3):148-151) can beadapted for use in the methods set forth herein. In certain embodiments,a CBE can comprise a fusion between a catalytically inactive Cas9(dCas9) RNA dependent DNA binding protein fused to a cytidine deaminasewhich converts cytosine (C) to uridine (U) and selected guide RNAs,thereby effecting a C to T substitution; see Komor et al. (2016) Nature,533:420-424. In other embodiments, C to T substitutions are effectedwith Cas9 nickase [Cas9n(D10A)] fused to an improved cytidine deaminaseand optionally a bacteriophage Mu dsDNA (double-stranded DNA)end-binding protein Gam; see Komor et al., Sci Adv. 2017 August;3(8):eaao4774. In other embodiments, adenine base editors (ABEs)comprising an adenine deaminase fused to catalytically inactive Cas9(dCas9) or a Cas9 D10A nickase can be used to convert A/T base pairs toG/C base pairs in genomic DNA (Gaudelli et al., (2017) Nature551(7681):464-471.

In certain embodiments, zinc finger nucleases or zinc finger nickasescan also be used in the methods provided herein. Zinc-finger nucleasesare site-specific endonucleases comprising two protein domains: aDNA-binding domain, comprising a plurality of individual zinc fingerrepeats that each recognize between 9 and 18 base pairs, and aDNA-cleavage domain that comprises a nuclease domain (typically Fokl).The cleavage domain dimerizes in order to cleave DNA; therefore, a pairof ZFNs are required to target non-palindromic target polynucleotides.In certain embodiments, zinc finger nuclease and zinc finger nickasedesign methods which have been described (Urnov et al. (2010) NatureRev. Genet., 11:636-646; Mohanta et al. (2017) Genes vol. 8, 12: 399;Ramirez et al. Nucleic Acids Res. (2012); 40(12): 5560-5568; Liu et al.(2013) Nature Communications, 4: 2565) can be adapted for use in themethods set forth herein. The zinc finger binding domains of the zincfinger nuclease or nickase provide specificity and can be engineered tospecifically recognize any desired target DNA sequence. The zinc fingerDNA binding domains are derived from the DNA-binding domain of a largeclass of eukaryotic transcription factors called zinc finger proteins(ZFPs). The DNA-binding domain of ZFPs typically contains a tandem arrayof at least three zinc “fingers” each recognizing a specific triplet ofDNA. A number of strategies can be used to design the bindingspecificity of the zinc finger binding domain. One approach, termed“modular assembly”, relies on the functional autonomy of individual zincfingers with DNA. In this approach, a given sequence is targeted byidentifying zinc fingers for each component triplet in the sequence andlinking them into a multifinger peptide. Several alternative strategiesfor designing zinc finger DNA binding domains have also been developed.These methods are designed to accommodate the ability of zinc fingers tocontact neighboring fingers as well as nucleotide bases outside theirtarget triplet. Typically, the engineered zinc finger DNA binding domainhas a novel binding specificity, compared to a naturally-occurring zincfinger protein. Engineering methods include, for example, rationaldesign and various types of selection. Rational design includes, forexample, the use of databases of triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, e.g., U.S. Pat. Nos. 6,453,242 and6,534,261, both incorporated herein by reference in their entirety.Exemplary selection methods (e.g., phage display and yeast two-hybridsystems) can be adapted for use in the methods described herein. Inaddition, enhancement of binding specificity for zinc finger bindingdomains has been described in U.S. Pat. No. 6,794,136, incorporatedherein by reference in its entirety. In addition, individual zinc fingerdomains may be linked together using any suitable linker sequences.Examples of linker sequences are publicly known, e.g., see U.S. Pat.Nos. 6,479,626; 6,903,185; and 7,153,949, incorporated herein byreference in their entirety. The nucleic acid cleavage domain isnon-specific and is typically a restriction endonuclease, such as Fokl.This endonuclease must dimerize to cleave DNA. Thus, cleavage by Fokl aspart of a ZFN requires two adjacent and independent binding events,which must occur in both the correct orientation and with appropriatespacing to permit dimer formation. The requirement for two DNA bindingevents enables more specific targeting of long and potentially uniquerecognition sites. Fokl variants with enhanced activities have beendescribed and can be adapted for use in the methods described herein;see, e.g., Guo et al. (2010) J. Mol. Biol., 400:96-107.

Transcription activator like effectors (TALEs) are proteins secreted bycertain Xanthomonas species to modulate gene expression in host plantsand to facilitate the colonization by and survival of the bacterium.TALEs act as transcription factors and modulate expression of resistancegenes in the plants. Recent studies of TALEs have revealed the codelinking the repetitive region of TALEs with their target DNA-bindingsites. TALEs comprise a highly conserved and repetitive regionconsisting of tandem repeats of mostly 33 or 34 amino acid segments. Therepeat monomers differ from each other mainly at amino acid positions 12and 13. A strong correlation between unique pairs of amino acids atpositions 12 and 13 and the corresponding nucleotide in the TALE-bindingsite has been found. The simple relationship between amino acid sequenceand DNA recognition of the TALE binding domain allows for the design ofDNA binding domains of any desired specificity. TALEs can be linked to anon-specific DNA cleavage domain to prepare genome editing proteins,referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs,a restriction endonuclease, such as Fokl, can be conveniently used.Methods for use of TALENs in plants have been described and can beadapted for use in the methods described herein, see Mahfouz et al.(2011) Proc. Natl. Acad. Sci. USA, 108:2623-2628; Mahfouz (2011) GMCrops, 2:99-103; and Mohanta et al. (2017) Genes vol. 8, 12: 399). TALEnickases have also been described and can be adapted for use in methodsdescribed herein (Wu et al.; Biochem Biophys Res Commun. (2014);446(1):261-6; Luo et al; Scientific Reports 6, Article number: 20657(2016)).

Embodiments of the donor DNA template molecule having a sequence that isintegrated at the site of at least one double-strand break (DSB) in agenome include double-stranded DNA, a single-stranded DNA, asingle-stranded DNA/RNA hybrid, and a double-stranded DNA/RNA hybrid. Inembodiments, a donor DNA template molecule that is a double-stranded(e.g., a dsDNA or dsDNA/RNA hybrid) molecule is provided directly to theplant protoplast or plant cell in the form of a double-stranded DNA or adouble-stranded DNA/RNA hybrid, or as two single-stranded DNA (ssDNA)molecules that are capable of hybridizing to form dsDNA, or as asingle-stranded DNA molecule and a single-stranded RNA (ssRNA) moleculethat are capable of hybridizing to form a double-stranded DNA/RNAhybrid; that is to say, the double-stranded polynucleotide molecule isnot provided indirectly, for example, by expression in the cell of adsDNA encoded by a plasmid or other vector. In various non-limitingembodiments of the method, the donor DNA template molecule that isintegrated (or that has a sequence that is integrated) at the site of atleast one double-strand break (DSB) in a genome is double-stranded andblunt-ended; in other embodiments the donor DNA template molecule isdouble-stranded and has an overhang or “sticky end” consisting ofunpaired nucleotides (e.g., 1, 2, 3, 4, 5, or 6 unpaired nucleotides) atone terminus or both termini. In an embodiment, the DSB in the genomehas no unpaired nucleotides at the cleavage site, and the donor DNAtemplate molecule that is integrated (or that has a sequence that isintegrated) at the site of the DSB is a blunt-ended double-stranded DNAor blunt-ended double-stranded DNA/RNA hybrid molecule, or alternativelyis a single-stranded DNA or a single-stranded DNA/RNA hybrid molecule.In another embodiment, the DSB in the genome has one or more unpairednucleotides at one or both sides of the cleavage site, and the donor DNAtemplate molecule that is integrated (or that has a sequence that isintegrated) at the site of the DSB is a double-stranded DNA ordouble-stranded DNA/RNA hybrid molecule with an overhang or “sticky end”consisting of unpaired nucleotides at one or both termini, oralternatively is a single-stranded DNA or a single-stranded DNA/RNAhybrid molecule; in embodiments, the donor DNA template molecule DSB isa double-stranded DNA or double-stranded DNA/RNA hybrid molecule thatincludes an overhang at one or at both termini, wherein the overhangconsists of the same number of unpaired nucleotides as the number ofunpaired nucleotides created at the site of a DSB by a nuclease thatcuts in an off-set fashion (e.g., where a Cas12 nuclease effects anoff-set DSB with 5-nucleotide overhangs in the genomic sequence, thedonor DNA template molecule that is to be integrated (or that has asequence that is to be integrated) at the site of the DSB isdouble-stranded and has 5 unpaired nucleotides at one or both termini).In certain embodiments, one or both termini of the donor DNA templatemolecule contain no regions of sequence homology (identity orcomplementarity) to genomic regions flanking the DSB; that is to say,one or both termini of the donor DNA template molecule contain noregions of sequence that is sufficiently complementary to permithybridization to genomic regions immediately adjacent to the location ofthe DSB. In embodiments, the donor DNA template molecule contains nohomology to the locus of the DSB, that is to say, the donor DNA templatemolecule contains no nucleotide sequence that is sufficientlycomplementary to permit hybridization to genomic regions immediatelyadjacent to the location of the DSB. In embodiments, the donor DNAtemplate molecule is at least partially double-stranded and includes2-20 base-pairs, e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 base-pairs; in embodiments, the donor DNA templatemolecule is double-stranded and blunt-ended and consists of 2-20base-pairs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 base-pairs; in other embodiments, the donor DNAtemplate molecule is double-stranded and includes 2-20 base-pairs, e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20base-pairs and in addition has at least one overhang or “sticky end”consisting of at least one additional, unpaired nucleotide at one or atboth termini. In an embodiment, the donor DNA template molecule that isintegrated (or that has a sequence that is integrated) at the site of atleast one double-strand break (DSB) in a genome is a blunt-endeddouble-stranded DNA or a blunt-ended double-stranded DNA/RNA hybridmolecule of about 18 to about 300 base-pairs, or about 20 to about 200base-pairs, or about 30 to about 100 base-pairs, and having at least onephosphorothioate bond between adjacent nucleotides at a 5′ end, 3′ end,or both 5′ and 3′ ends. In embodiments, the donor DNA template moleculeincludes single strands of at least 11, at least 18, at least 20, atleast 30, at least 40, at least 60, at least 80, at least 100, at least120, at least 140, at least 160, at least 180, at least 200, at least240, at about 280, or at least 320 nucleotides. In embodiments, thedonor DNA template molecule has a length of at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, or at least 11 base-pairs if double-stranded (or nucleotidesif single-stranded), or between about 2 to about 320 base-pairs ifdouble-stranded (or nucleotides if single-stranded), or between about 2to about 500 base-pairs if double-stranded (or nucleotides ifsingle-stranded), or between about 5 to about 500 base-pairs ifdouble-stranded (or nucleotides if single-stranded), or between about 5to about 300 base-pairs if double-stranded (or nucleotides ifsingle-stranded), or between about 11 to about 300 base-pairs ifdouble-stranded (or nucleotides if single-stranded), or about 18 toabout 300 base-pairs if double-stranded (or nucleotides ifsingle-stranded), or between about 30 to about 100 base-pairs ifdouble-stranded (or nucleotides if single-stranded). In embodiments, thedonor DNA template molecule includes chemically modified nucleotides(see, e.g., the various modifications of internucleotide linkages,bases, and sugars described in Verma and Eckstein (1998) Annu. Rev.Biochem., 67:99-134); in embodiments, the naturally occurringphosphodiester backbone of the donor DNA template molecule is partiallyor completely modified with phosphorothioate, phosphorodithioate, ormethylphosphonate internucleotide linkage modifications, or the donorDNA template molecule includes modified nucleoside bases or modifiedsugars, or the donor DNA template molecule is labelled with afluorescent moiety (e.g., fluorescein or rhodamine or a fluorescentnucleoside analogue) or other detectable label (e.g., biotin or anisotope). In another embodiment, the donor DNA template moleculecontains secondary structure that provides stability or acts as anaptamer. Other related embodiments include double-stranded DNA/RNAhybrid molecules, single-stranded DNA/RNA hybrid donor molecules, andsingle-stranded DNA donor molecules (including single-stranded,chemically modified DNA donor molecules), which in analogous proceduresare integrated (or have a sequence that is integrated) at the site of adouble-strand break.

Donor DNA template molecules used in the methods provided herein includeDNA molecules comprising, from 5′ to 3′, a first homology arm, areplacement DNA, and a second homology arm, wherein the homology armscontaining sequences that are partially or completely homologous togenomic DNA (gDNA) sequences flanking a target site-specificendonuclease cleavage site in the gDNA. In certain embodiments, thereplacement DNA can comprise an insertion, deletion, or substitution of1 or more DNA base pairs relative to the target gDNA. In an embodiment,the donor DNA template molecule is double-stranded and perfectlybase-paired through all or most of its length, with the possibleexception of any unpaired nucleotides at either terminus or bothtermini. In another embodiment, the donor DNA template molecule isdouble-stranded and includes one or more non-terminal mismatches ornon-terminal unpaired nucleotides within the otherwise double-strandedduplex. In an embodiment, the donor DNA template molecule that isintegrated at the site of at least one double-strand break (DSB)includes between 2-20 nucleotides in one (if single-stranded) or in bothstrands (if double-stranded), e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleotides on one or on both strands,each of which can be base-paired to a nucleotide on the opposite strand(in the case of a perfectly base-paired double-stranded polynucleotidemolecule). Such donor DNA templates can be integrated in genomic DNAcontaining blunt and/or staggered double stranded DNA breaks byhomology-directed repair (HDR). In certain embodiments, a donor DNAtemplate homology arm can be about 20, 50, 100, 200, 400, or 600 toabout 800, or 1000 base pairs in length. In certain embodiments, a donorDNA template molecule can be delivered to a plant cell) in a circular(e.g., a plasmid or a viral vector including a geminivirus vector) or alinear DNA molecule. In certain embodiments, a circular or linear DNAmolecule that is used can comprise a modified donor DNA templatemolecule comprising, from 5′ to 3′, a first copy of the targetsequence-specific endonuclease cleavage site sequence, the firsthomology arm, the replacement DNA, the second homology arm, and a secondcopy of the target sequence-specific endonuclease cleavage sitesequence. Without seeking to be limited by theory, such modified donorDNA template molecules can be cleaved by the same sequence-specificendonuclease that is used to cleave the target site gDNA of theeukaryotic cell to release a donor DNA template molecule that canparticipate in HDR-mediated genome modification of the target editingsite in the plant cell genome. In certain embodiments, the donor DNAtemplate can comprise a linear DNA molecule comprising, from 5′ to 3′, acleaved target sequence-specific endonuclease cleavage site sequence,the first homology arm, the replacement DNA, the second homology arm,and a cleaved target sequence-specific endonuclease cleavage sitesequence. In certain embodiments, the cleaved target sequence-specificendonuclease sequence can comprise a blunt DNA end or a blunt DNA endthat can optionally comprise a 5′ phosphate group. In certainembodiments, the cleaved target sequence-specific endonuclease sequencecomprises a DNA end having a single-stranded 5′ or 3′ DNA overhang. Suchcleaved target sequence-specific endonuclease cleavage site sequencescan be produced by either cleaving an intact target sequence-specificendonuclease cleavage site sequence or by synthesizing a copy of thecleaved target sequence-specific endonuclease cleavage site sequence.Donor DNA templates can be synthesized either chemically orenzymatically (e.g., in a polymerase chain reaction (PCR)).

Various treatments are useful in delivery of gene editing moleculesand/or other molecules to a plant cell. In certain embodiments, one ormore treatments is employed to deliver the gene editing or othermolecules (e.g., comprising a polynucleotide, polypeptide or combinationthereof) into a eukaryotic or plant cell, e.g., through barriers such asa cell wall, a plasma membrane, a nuclear envelope, and/or other lipidbilayer. In certain embodiments, a polynucleotide-, polypeptide-, orRNP-containing composition comprising the molecules are delivereddirectly, for example by direct contact of the composition with a plantcell. Aforementioned compositions can be provided in the form of aliquid, a solution, a suspension, an emulsion, a reverse emulsion, acolloid, a dispersion, a gel, liposomes, micelles, an injectablematerial, an aerosol, a solid, a powder, a particulate, a nanoparticle,or a combination thereof can be applied directly to a plant, plant part,plant cell, or plant explant (e.g., through abrasion or puncture orotherwise disruption of the cell wall or cell membrane, by spraying ordipping or soaking or otherwise directly contacting, by microinjection).For example, a plant cell or plant protoplast is soaked in a liquidgenome editing molecule-containing composition, whereby the agent isdelivered to the plant cell. In certain embodiments, theagent-containing composition is delivered using negative or positivepressure, for example, using vacuum infiltration or application ofhydrodynamic or fluid pressure. In certain embodiments, theagent-containing composition is introduced into a plant cell or plantprotoplast, e.g., by microinjection or by disruption or deformation ofthe cell wall or cell membrane, for example by physical treatments suchas by application of negative or positive pressure, shear forces, ortreatment with a chemical or physical delivery agent such assurfactants, liposomes, or nanoparticles; see, e.g., delivery ofmaterials to cells employing microfluidic flow through a cell-deformingconstriction as described in US Published Patent Application2014/0287509, incorporated by reference in its entirety herein. Othertechniques useful for delivering the agent-containing composition to aeukaryotic cell, plant cell or plant protoplast include: ultrasound orsonication; vibration, friction, shear stress, vortexing, cavitation;centrifugation or application of mechanical force; mechanical cell wallor cell membrane deformation or breakage; enzymatic cell wall or cellmembrane breakage or permeabilization; abrasion or mechanicalscarification (e.g., abrasion with carborundum or other particulateabrasive or scarification with a file or sandpaper) or chemicalscarification (e.g., treatment with an acid or caustic agent); andelectroporation. In certain embodiments, the agent-containingcomposition is provided by bacterially mediated (e.g., Agrobacteriumsp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobiumsp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cellor plant protoplast with a polynucleotide encoding the genome editingmolecules (e.g., RNA dependent DNA endonuclease, RNA dependent DNAbinding protein, RNA dependent nickase, ABE, or CBE, and/or guide RNA);see, e.g., Broothaerts et al. (2005) Nature, 433:629-633). Any of thesetechniques or a combination thereof are alternatively employed on theplant explant, plant part or tissue or intact plant (or seed) from whicha plant cell is optionally subsequently obtained or isolated; in certainembodiments, the agent-containing composition is delivered in a separatestep after the plant cell has been isolated.

In some embodiments, one or more polynucleotides or vectors drivingexpression of one or more genome editing molecules or trait-conferringgenes (e.g.; herbicide tolerance, insect resistance, and/or malesterility) are introduced into a plant cell. In certain embodiments, apolynucleotide vector comprises a regulatory element such as a promoteroperably linked to one or more polynucleotides encoding genome editingmolecules and/or trait-conferring genes. In such embodiments, expressionof these polynucleotides can be controlled by selection of theappropriate promoter, particularly promoters functional in a eukaryoticcell (e.g., plant cell); useful promoters include constitutive,conditional, inducible, and temporally or spatially specific promoters(e.g., a tissue specific promoter, a developmentally regulated promoter,or a cell cycle regulated promoter). Developmentally regulated promotersthat can be used in plant cells include Phospholipid Transfer Protein(PLTP), fructose-1,6-bisphosphatase protein, NAD(P)-bindingRossmann-Fold protein, adipocyte plasma membrane-associated protein-likeprotein, Rieske [2Fe-2S] iron-sulfur domain protein, chlororespiratoryreduction 6 protein, D-glycerate 3-kinase, chloroplastic-like protein,chlorophyll a-b binding protein 7, chloroplastic-like protein,ultraviolet-B-repressible protein, Soul heme-binding family protein,Photosystem I reaction center subunit psi-N protein, and short-chaindehydrogenase/reductase protein that are disclosed in US PatentApplication Publication No. 20170121722, which is incorporated herein byreference in its entirety and specifically with respect to suchdisclosure. In certain embodiments, the promoter is operably linked tonucleotide sequences encoding multiple guide RNAs, wherein the sequencesencoding guide RNAs are separated by a cleavage site such as anucleotide sequence encoding a microRNA recognition/cleavage site or aself-cleaving ribozyme (see, e.g., Ferré-D'Amaré and Scott (2014) ColdSpring Harbor Perspectives Biol., 2:a003574). In certain embodiments,the promoter is an RNA polymerase III promoter operably linked to anucleotide sequence encoding one or more guide RNAs. In certainembodiments, the RNA polymerase III promoter is a plant U6 spliceosomalRNA promoter, which can be native to the genome of the plant cell orfrom a different species, e.g., a U6 promoter from maize, tomato, orsoybean such as those disclosed U.S. Patent Application Publication2017/0166912, or a homologue thereof; in an example, such a promoter isoperably linked to DNA sequence encoding a first RNA molecule includinga Cas12a gRNA followed by an operably linked and suitable 3′ elementsuch as a U6 poly-T terminator. In another embodiment, the RNApolymerase III promoter is a plant U3, 7SL (signal recognition particleRNA), U2, or U5 promoter, or chimerics thereof, e.g., as described inU.S. Patent Application Publication 20170166912. In certain embodiments,the promoter operably linked to one or more polynucleotides is aconstitutive promoter that drives gene expression in eukaryotic cells(e.g., plant cells). In certain embodiments, the promoter drives geneexpression in the nucleus or in an organelle such as a chloroplast ormitochondrion. Examples of constitutive promoters for use in plantsinclude a CaMV 35S promoter as disclosed in U.S. Pat. Nos. 5,858,742 and5,322,938, a rice actin promoter as disclosed in U.S. Pat. No.5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S.Pat. No. 7,151,204, and the nopaline synthase (NOS) and octopinesynthase (OCS) promoters from Agrobacterium tumefaciens. In certainembodiments, the promoter operably linked to one or more polynucleotidesencoding elements of a genome-editing system is a promoter from figwortmosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase(PPDK) promoter, which is active in photosynthetic tissues. Othercontemplated promoters include cell-specific or tissue-specific ordevelopmentally regulated promoters, for example, a promoter that limitsthe expression of the nucleic acid targeting system to germline orreproductive cells (e.g., promoters of genes encoding DNA ligases,recombinases, replicases, or other genes specifically expressed ingermline or reproductive cells). In certain embodiments, the genomealteration is limited only to those cells from which DNA is inherited insubsequent generations, which is advantageous where it is desirable thatexpression of the genome-editing system be limited in order to avoidgenotoxicity or other unwanted effects. All of the patent publicationsreferenced in this paragraph are incorporated herein by reference intheir entirety.

Expression vectors or polynucleotides provided herein may contain a DNAsegment near the 3′ end of an expression cassette that acts as a signalto terminate transcription and directs polyadenylation of the resultantmRNA and may also support promoter activity. Such a 3′ element iscommonly referred to as a “3′-untranslated region” or “3′-UTR” or a“polyadenylation signal.” In some cases, plant gene-based 3′ elements(or terminators) consist of both the 3′-UTR and downstreamnon-transcribed sequence (Nuccio et al., 2015). Useful 3′ elementsinclude: Agrobacterium tumefaciens nos 3′, tml 3′, tmr 3′, tms 3′, ocs3′, and tr7 3′ elements disclosed in U.S. Pat. No. 6,090,627,incorporated herein by reference, and 3′ elements from plant genes suchas the heat shock protein 17, ubiquitin, and fructose-1,6-biphosphatasegenes from wheat (Triticum aestivum), and the glutelin, lactatedehydrogenase, and beta-tubulin genes from rice (Oryza sativa),disclosed in US Patent Application Publication 2002/0192813 A1. All ofthe patent publications referenced in this paragraph are incorporatedherein by reference in their entireties.

In certain embodiments, the plant cells can comprise haploid, diploid,or polyploid plant cells or plant protoplasts, for example, thoseobtained from a haploid, diploid, or polyploid plant, plant part ortissue, or callus. In certain embodiments, plant cells in culture (orthe regenerated plant, progeny seed, and progeny plant) are haploid orcan be induced to become haploid; techniques for making and usinghaploid plants and plant cells are known in the art, see, e.g., methodsfor generating haploids in Arabidopsis thaliana by crossing of awild-type strain to a haploid-inducing strain that expresses alteredforms of the centromere-specific histone CENH3, as described byMaruthachalam and Chan in “How to make haploid Arabidopsis thaliana”,protocol available atwww[dot]openwetware[dot]org/images/d/d3/Haploid_Arabidopsis_protocol[dot]pdf;(Ravi et al. (2014) Nature Communications, 5:5334, doi:10.1038/ncomms6334). Haploids can also be obtained in a wide variety ofmonocot plants (e.g., maize, wheat, rice, sorghum, barley) or dicotplants (e.g., soybean, Brassica sp. including canola, cotton, tomato) bycrossing a plant comprising a mutated CENH3 gene with a wildtype diploidplant to generate haploid progeny as disclosed in U.S. Pat. No.9,215,849, which is incorporated herein by reference in its entirety.Haploid-inducing maize lines that can be used to obtain haploid maizeplants and/or cells include Stock 6, MHI (Moldovian Haploid Inducer),indeterminate gametophyte (ig) mutation, KEMS, RWK, ZEM, ZMS, KMS, andwell as transgenic haploid inducer lines disclosed in U.S. Pat. No.9,677,082, which is incorporated herein by reference in its entirety.Examples of haploid cells include but are not limited to plant cellsobtained from haploid plants and plant cells obtained from reproductivetissues, e.g., from flowers, developing flowers or flower buds, ovaries,ovules, megaspores, anthers, pollen, megagametophyte, and microspores.In certain embodiments where the plant cell or plant protoplast ishaploid, the genetic complement can be doubled by chromosome doubling(e.g., by spontaneous chromosomal doubling by meiotic non-reduction, orby using a chromosome doubling agent such as colchicine, oryzalin,trifluralin, pronamide, nitrous oxide gas, anti-microtubule herbicides,anti-microtubule agents, and mitotic inhibitors) in the plant cell orplant protoplast to produce a doubled haploid plant cell or plantprotoplast wherein the complement of genes or alleles is homozygous; yetother embodiments include regeneration of a doubled haploid plant fromthe doubled haploid plant cell or plant protoplast. Another embodimentis related to a hybrid plant having at least one parent plant that is adoubled haploid plant provided by this approach. Production of doubledhaploid plants provides homozygosity in one generation, instead ofrequiring several generations of self-crossing to obtain homozygousplants. The use of doubled haploids is advantageous in any situationwhere there is a desire to establish genetic purity (i.e. homozygosity)in the least possible time. Doubled haploid production can beparticularly advantageous in slow-growing plants or for producing hybridplants that are offspring of at least one doubled-haploid plant.

In certain embodiments, the plant cells used in the methods providedherein can include non-dividing cells. Such non-dividing cells caninclude plant cell protoplasts, plant cells subjected to one or more ofa genetic and/or pharmaceutically induced cell-cycle blockage, and thelike.

In certain embodiments, the plant cells in used in the methods providedherein can include dividing cells. Dividing cells can include thosecells found in various plant tissues including leaves, meristems, andembryos. These tissues include dividing cells from young maize leaf,meristems and scutellar tissue from about 8 or 10 to about 12 or 14 daysafter pollination (DAP) embryos. The isolation of maize embryos has beendescribed in several publications (Brettschneider, Becker, and Lorz1997; Leduc et al. 1996; Frame et al. 2011; K. Wang and Frame 2009). Incertain embodiments, basal leaf tissues (e.g., leaf tissues locatedabout 0 to 3 cm from the ligule of a maize plant; Kirienko, Luo, andSylvester 2012) are targeted for HDR-mediated gene editing. Methods forobtaining regenerable plant structures and regenerating plants from theNHEJ-, MMEJ-, or HDR-mediated gene editing of plant cells providedherein can be adapted from methods disclosed in US Patent ApplicationPublication No. 20170121722, which is incorporated herein by referencein its entirety and specifically with respect to such disclosure. Incertain embodiments, single plant cells subjected to the HDR-mediatedgene editing will give rise to single regenerable plant structures. Incertain embodiments, the single regenerable plant cell structure canform from a single cell on, or within, an explant that has beensubjected to the NHEJ-, MMEJ-, or HDR-mediated gene editing.

In some embodiments, methods provided herein can include the additionalstep of growing or regenerating a plant from a plant cell that had beensubjected to the improved HDR-mediated gene editing or from aregenerable plant structure obtained from that plant cell. In certainembodiments, the plant can further comprise an inserted transgene, atarget gene edit, or genome edit as provided by the methods andcompositions disclosed herein. In certain embodiments, callus isproduced from the plant cell, and plantlets and plants produced fromsuch callus. In other embodiments, whole seedlings or plants are growndirectly from the plant cell without a callus stage. Thus, additionalrelated aspects are directed to whole seedlings and plants grown orregenerated from the plant cell or plant protoplast having a target geneedit or genome edit, as well as the seeds of such plants. In certainembodiments wherein the plant cell or plant protoplast is subjected togenetic modification (for example, genome editing by means of, e.g., anRdDe), the grown or regenerated plant exhibits a phenotype associatedwith the genetic modification. In certain embodiments, the grown orregenerated plant includes in its genome two or more genetic orepigenetic modifications that in combination provide at least onephenotype of interest. In certain embodiments, a heterogeneouspopulation of plant cells having a target gene edit or genome edit, atleast some of which include at least one genetic or epigeneticmodification, is provided by the method; related aspects include a planthaving a phenotype of interest associated with the genetic or epigeneticmodification, provided by either regeneration of a plant having thephenotype of interest from a plant cell or plant protoplast selectedfrom the heterogeneous population of plant cells having a target gene orgenome edit, or by selection of a plant having the phenotype of interestfrom a heterogeneous population of plants grown or regenerated from thepopulation of plant cells having a targeted genetic edit or genome edit.Examples of phenotypes of interest include herbicide resistance,improved tolerance of abiotic stress (e.g., tolerance of temperatureextremes, drought, or salt) or biotic stress (e.g., resistance tonematode, bacterial, or fungal pathogens), improved utilization ofnutrients or water, modified lipid, carbohydrate, or proteincomposition, improved flavor or appearance, improved storagecharacteristics (e.g., resistance to bruising, browning, or softening),increased yield, altered morphology (e.g., floral architecture or color,plant height, branching, root structure). In an embodiment, aheterogeneous population of plant cells having a target gene edit orgenome edit (or seedlings or plants grown or regenerated therefrom) isexposed to conditions permitting expression of the phenotype ofinterest; e.g., selection for herbicide resistance can include exposingthe population of plant cells having a target gene edit or genome edit(or seedlings or plants grown or regenerated therefrom) to an amount ofherbicide or other substance that inhibits growth or is toxic, allowingidentification and selection of those resistant plant cells (orseedlings or plants) that survive treatment. Methods for obtainingregenerable plant structures and regenerating plants from plant cells orregenerable plant structures can be adapted from published procedures(Roest and Gilissen, Acta Bot. Neerl., 1989, 38(1), 1-23; Bhaskaran andSmith, Crop Sci. 30(6):1328-1337; Ikeuchi et al., Development, 2016,143: 1442-1451). Methods for obtaining regenerable plant structures andregenerating plants from plant cells or regenerable plant structures canalso be adapted from US Patent Application Publication No. 20170121722,which is incorporated herein by reference in its entirety andspecifically with respect to such disclosure. Also provided areheterogeneous or homogeneous populations of such plants or parts thereof(e.g., seeds), succeeding generations or seeds of such plants grown orregenerated from the plant cells or plant protoplasts, having a targetgene edit or genome edit. Additional related aspects include a hybridplant provided by crossing a first plant grown or regenerated from aplant cell or plant protoplast having a target gene edit or genome editand having at least one genetic or epigenetic modification, with asecond plant, wherein the hybrid plant contains the genetic orepigenetic modification; also contemplated is seed produced by thehybrid plant. Also envisioned as related aspects are progeny seed andprogeny plants, including hybrid seed and hybrid plants, having theregenerated plant as a parent or ancestor. The plant cells andderivative plants and seeds disclosed herein can be used for variouspurposes useful to the consumer or grower. In other embodiments,processed products are made from the plant or its seeds, including: (a)corn, soy, cotton, or canola seed meal (defatted or non-defatted); (b)extracted proteins, oils, sugars, and starches; (c) fermentationproducts; (d) animal feed or human food products (e.g., feed and foodcomprising corn, soy, cotton, or canola seed meal (defatted ornon-defatted) and other ingredients (e.g., other cereal grains, otherseed meal, other protein meal, other oil, other starch, other sugar, abinder, a preservative, a humectant, a vitamin, and/or mineral; (e) apharmaceutical; (f) raw or processed biomass (e.g., cellulosic and/orlignocellulosic material); and (g) various industrial products.

Embodiments

Various embodiments of the plants, genomes, methods, biological samples,and other compositions described herein are set forth in the followingsets of numbered embodiments.

1. An edited transgenic plant genome comprising a first modifiedtransgenic locus that comprises:

(i) a first originator guide RNA recognition site (OgRRS) comprising aprotospacer adjacent motif (PAM) site operably linked to a guide RNAhybridization site, wherein the OgRRS is located in transgenic DNA, innon-transgenic plant genomic DNA, or in a combination thereof in a firstDNA junction polynucleotide of the first modified transgenic locus; and(ii) a first cognate guide RNA recognition site (CgRRS) comprising aprotospacer adjacent motif (PAM) site operably linked to a guide RNAhybridization site located in a second DNA junction polynucleotide ofthe first modified transgenic locus, wherein the CgRRS is absent fromtransgenic plant genomes comprising a first original transgenic locusthat is unmodified and wherein the OgRRS and the CgRRS can hybridize toone first guide RNA (gRNA).

2. The edited transgenic plant genome of embodiment 1, wherein the guideRNA hybridization site of the OgRRS and the CgRRS comprise at least 18nucleotides of identical DNA sequence.

3. The edited transgenic plant genome of embodiment 1, wherein the OgRRSand the CgRRS can be cleaved by the same RNA dependent DNA endonuclease(RdDe) when the OgRRS and the CgRRS are hybridized to the one firstgRNA.

4. The edited transgenic plant genome of embodiment 3, wherein the RdDeis a class 2 type II or class 2 type V RdDe.

5. The edited transgenic plant genome of embodiment 1, wherein the PAMsite in the OgRRS and the PAM site in the CgRRS comprise distinct PAMsequences that are recognized by the same RdDe when the OgRRS and theCgRRS are hybridized to the one gRNA.

6. The edited transgenic plant genome of any one of embodiments 1 to 5,wherein the OgRRS is located in non-transgenic DNA, in non-transgenicDNA and within about 1000, 750, 500, 250, 100, or 50 base pairs (bp) oftransgenic DNA, or within transgenic DNA of the first DNA junctionpolynucleotide; and/or wherein the CgRRS is located within innon-transgenic DNA, in non-transgenic DNA and about 1000, 750, 500, 250,100, or 50 bp of transgenic DNA in the second DNA junctionpolynucleotide, or within transgenic DNA of the second DNA junctionpolynucleotide.

7. The edited transgenic plant genome of any one of embodiments 1 to 6,wherein the edited transgenic plant genome further comprises a secondmodified transgenic locus that comprises:

(i) a second OgRRS located in transgenic DNA, in non-transgenic plantgenomic DNA, or in a combination thereof in a first DNA junctionpolynucleotide of the second modified transgenic locus; and(ii) a second CgRRS located in a second DNA junction polynucleotide ofthe second modified transgenic locus, wherein the CgRRS is absent fromunedited transgenic plant genomes comprising a second originaltransgenic locus, optionally wherein the second OgRRS and the secondCgRRS can hybridize to one second guide RNA (gRNA) and optionallywherein the second guide RNA is not complementary to the first OgRRS andthe first CgRRS.

8. The edited transgenic plant genome of any one of embodiments 1 to 7,wherein the first and/or second modified transgenic locus comprises atleast one modification of a Bt11, DAS-59122-7, DP-4114, GA21, MON810,MON87411, MON87427, MON88017, MON89034, MIR162, MIR604, NK603,SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038,MON863, MON87403, MON87403, MON87419, MON87460, MZHG0JG, MZIR098,VCO-Ø1981-5, 98140, or TC1507 original transgenic locus in a transgeniccorn plant genome, wherein the modification comprises the CgRRS in thesecond DNA junction polynucleotide of the first transgenic locus andoptionally the CgRRS in the second DNA junction polynucleotide of thesecond modified transgenic locus, and optionally wherein themodification further comprises a deletion of at least one selectablemarker gene and/or non-essential DNA in the original transgenic locus.

9. The edited transgenic plant genome of any one of embodiments 1 to 7,wherein the first and/or second modified transgenic locus comprises amodification of an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 originaltransgenic locus in a transgenic soybean plant genome, wherein themodification comprises the CgRRS in the second DNA junctionpolynucleotide of the first transgenic locus and optionally the CgRRS inthe second DNA junction polynucleotide of the second modified transgeniclocus, and wherein the modification optionally further comprises adeletion of at least one selectable marker gene and/or non-essential DNAin the original transgenic locus.

10. The edited transgenic plant genome of any one of embodiments 1 to 7,wherein the first and/or second modified transgenic locus comprises atleast one modification of a DAS-21023-5, DAS-24236-5, COT102,LLcotton25, MON15985, MON88701, or MON88913 original transgenic locus ina transgenic cotton plant genome, wherein the modification comprises theCgRRS in the second DNA junction polynucleotide of the first transgeniclocus and optionally the CgRRS in the second DNA junction polynucleotideof the second modified transgenic locus, and wherein the modificationoptionally further comprises a deletion of at least one selectablemarker gene and/or non-essential DNA in the original transgenic locus.

11. The edited transgenic plant genome of any one of embodiments 1 to 7,wherein the first and/or second modified transgenic locus comprises amodification of an GT73, HCN28, MON88302, or MS8 original transgeniclocus in a transgenic canola plant genome, wherein the modificationcomprises the CgRRS in the second DNA junction polynucleotide of thefirst transgenic locus and optionally the CgRRS in the second DNAjunction polynucleotide of the second modified transgenic locus, andwherein the modification optionally further comprises a deletion of atleast one selectable marker gene and/or non-essential DNA in theoriginal transgenic locus.

12. The edited transgenic plant genome of any one of embodiments to 1 to11, wherein the first and/or second modified transgenic locus lacks aselectable marker transgene which confers resistance to an antibiotic,tolerance to an herbicide, or an ability to grow on a specific carbonsource, optionally wherein the selectable marker transgene was presentin the original transgenic locus and/or wherein the specific carbonsource is optionally mannose.

13. The edited transgenic plant genome of any one of embodiments 1 to12, wherein: (i) the first CgRRS is located in non-transgenic plantgenomic DNA of the second DNA junction polynucleotide of the firstmodified transgenic locus; or (ii) the first CgRRS is located intransgenic DNA of the second DNA junction polynucleotide of the firstmodified transgenic locus.

14. The edited transgenic plant genome of embodiment 13, wherein theCgRRS is located in the non-transgenic plant genomic DNA and comprises asequence having 50% to 70% sequence identity to the non-transgenic plantgenomic DNA located at the same chromosomal location in the originaltransgenic locus.

15. The edited transgenic plant genome of any one of embodiments 1 to14, wherein the first and/or second transgenic locus further comprise asecond introduced transgene or optionally wherein the second introducedtransgene is integrated at a site in the modified transgenic locus whichwas occupied by a selectable marker transgene in the original transgeniclocus.

16. The edited transgenic plant genome of any one of embodiments 1 to15, wherein the genome further comprises a targeted genetic change.

17. A transgenic plant cell comprising the edited transgenic plantgenome of any one of embodiments 1 to 16.

18. A transgenic plant comprising the transgenic plant genome of any oneof embodiments 1 to 16.

19. A transgenic plant part comprising the edited transgenic plantgenome of any one of embodiments 1 to 16.

20. The transgenic plant part of embodiment 19, wherein the part is aseed, leaf, tuber, stem, root, or boll.

21. A method for obtaining a bulked population of inbred seed forcommercial seed production comprising selfing the transgenic plant ofembodiment 18 and harvesting seed from the selfed crop plants.

22. A method of obtaining hybrid crop seed comprising crossing a firstcrop plant comprising the transgenic plant of embodiment 18 to a secondcrop plant and harvesting seed from the cross.

23. The method of embodiment 22 wherein the first crop plant and thesecond crop plant are in distinct heterotic groups.

24. The method of embodiment 22 or 23, wherein either the first orsecond crop plant are pollen recipients which have been rendered malesterile.

25. The method of any one of embodiments 22 to 24, wherein the cropplant is rendered male sterile by emasculation, cytoplasmic malesterility, a chemical hybridizing agent or system, a transgene, and/or amutation in an endogenous plant gene.

26. The method of any one of embodiments 22 to 25, further comprisingthe step of sowing the hybrid crop seed.

27. A DNA molecule comprising the cognate guide RNA recognition site(CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plantgenomic DNA flanking the CgRRS, wherein the transgenic DNA ornon-transgenic plant genomic DNA comprises DNA sequences of the secondDNA junction polynucleotide of the first modified transgenic locus setforth in any one of embodiments 1 to 11.

28. The DNA of embodiment 27, wherein the modified transgenic locus is aBt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017,MON89034, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138,DP-33121, HCEM485, LY038, MON863, MON87403, MON87403, MON87419,MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, or TC1507 transgeniclocus.

29. The DNA of embodiment 27, wherein the modified transgenic locus isan A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701,MON87708, MON89788, MST-FGØ72-3, and/or SYHT0H2 transgenic locus.

30. The DNA of embodiment 27 wherein the modified transgenic locus is:(i) a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701,and/or MON88913 transgenic locus and wherein the modificationsoptionally further comprise a deletion of at least one selectable markergene and/or non-essential DNA in the original transgenic locus; or (ii)wherein the modified transgenic locus is a GT73, HCN28, MON88302, or MS8transgenic locus.

31. The DNA of any one of embodiments 27 to 30, wherein the DNA ispurified or isolated.

32. A processed transgenic plant product containing the DNA of any oneof embodiments 27 to 30.

33. A biological sample containing the DNA of any one of embodiments 27to 30.

34. A nucleic acid marker adapted for detection of genomic DNA orfragments thereof comprising a cognate guide RNA recognition site(CgRRS) in, adjacent to, or operably linked to a DNA junctionpolynucleotide of a modified transgenic locus.

35. The nucleic acid marker of embodiment 34, comprising apolynucleotide of at least 18 nucleotides in length which spans DNAsequences comprising both the CgRRS and non-transgenic plant genomic DNAflanking either a telomere-proximal or a centromere proximal end of theCgRRS.

36. The nucleic acid marker of embodiment 34 or 35, wherein the markerfurther comprises a detectable label.

37. The nucleic acid marker of any one of embodiments 34 to 36, whereinthe CgRRS and non-transgenic plant genomic DNA are identical tosequences in a modified transgenic locus.

38. The nucleic acid marker of any one of embodiments 34 to 37, whereinthe modified transgenic locus is a Bt11, DAS-59122-7, DP-4114, GA21,MON810, MON87411, MON87427, MON88017, MIR162, MIR604, NK603,SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038,MON863, MON87403, MON87403, MON87419, MON87460, MZHG0JG, MZIR098,VCO-Ø1981-5, 98140, or TC1507 transgenic locus comprising the CgRRS,wherein the CgRRS is located in, adjacent to, or operably linked to aDNA junction polynucleotide of the modified transgenic locus.

39. The nucleic acid marker of any one of embodiments 34 to 37, whereinthe modified transgenic locus is a modified A5547-127, DAS44406-6,DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,MST-FGØ72-3, or SYHT0H2 transgenic locus comprising the CgRRS, whereinthe CgRRS is located in, adjacent to, or operably linked to a DNAjunction polynucleotide of the modified transgenic locus.

40. The nucleic acid marker of any one of embodiments 34 to 37, whereinthe modified transgenic locus is a DAS-21023-5, DAS-24236-5, COT102,LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locuscomprising the CgRRS, wherein the CgRRS is located in, adjacent to, oroperably linked to a DNA junction polynucleotide of the modifiedtransgenic locus.

41. The nucleic acid marker of any one of embodiments 34 to 37, whereinthe modified transgenic locus is a GT73, HCN28, MON88302, or MS8transgenic locus comprising the CgRRS, wherein the CgRRS is located in,adjacent to, or operably linked to a DNA junction polynucleotide of themodified transgenic locus.

42. A processed transgenic plant product obtained from the transgenicplant part of embodiment 19 or 20, wherein the processed plant productcontains a polynucleotide comprising a cognate guide RNA recognitionsite (CgRRS) and at least 10 bp of transgenic DNA or non-transgenicplant genomic DNA flanking the CgRRS, wherein the transgenic DNA ornon-transgenic plant genomic DNA comprises transgenic or non-transgenicplant genomic DNA sequences of the second DNA junction polynucleotide ofthe first modified transgenic locus.

43. A biological sample obtained from the transgenic plant cell ofembodiment 17, the transgenic plant of embodiment 18, or the transgenicplant part of embodiment 19 or 20, wherein the biological samplecontains one or more polynucleotide(s) comprising the a cognate guideRNA recognition site (CgRRS) and at least 10 bp of transgenic DNA ornon-transgenic plant genomic DNA flanking the CgRRS, wherein thetransgenic DNA or non-transgenic plant genomic DNA comprises transgenicor non-transgenic plant genomic DNA sequences of the second DNA junctionpolynucleotide of the first modified transgenic locus.

44. A method of detecting the edited transgenic plant genome of any oneof embodiments 1 to 16, comprising the step of detecting the presence ofa polynucleotide comprising one or more of said CgRRS.

45. The method of embodiment 44, wherein the polynucleotide is detectedby detecting a single nucleotide polymorphism (SNP) in the CgRRS that ispresent in the modified transgenic locus but absent in the originaltransgenic locus.

46. The method of embodiment 44 or 45, wherein the edited transgenicplant genome is detected in a transgenic plant cell, a transgenic plantpart, a transgenic plant, a processed transgenic plant product, or abiological sample.

47. A method of obtaining an edited transgenic plant genome comprising amodified transgenic locus comprising the step of introducing a cognateguide RNA recognition site (CgRRS) in a DNA junction polynucleotide ofan original transgenic locus, wherein the CgRRS is in, adjacent to, oroperably linked to a DNA junction polynucleotide of the modifiedtransgenic locus.

48. The method of embodiment 47, wherein the CgRRS is located innon-transgenic plant genomic DNA of the DNA junction polynucleotide ofthe modified transgenic locus and optionally wherein the CgRRS comprisesa sequence having 50% to 70% sequence identity to the non-transgenicplant genomic DNA located at the same chromosomal location in theoriginal transgenic locus.

49. The method of embodiment 47 or 48, wherein the CgRRS is introducedby:

(a) contacting the original transgenic locus with: (i) an RNA dependentDNA endonuclease (RdDe) or RdDe nickase; and a guide RNA comprising anRNA equivalent of the DNA located immediately 5′ or 3′ to an originalPAM site located within the DNA junction polynucleotide of the originaltransgenic locus; or (ii) a guide RNA comprising an RNA equivalent ofthe DNA located immediately 5′ or 3′ to an original PAM site locatedwithin the DNA junction polynucleotide of the original transgenic locus;and (iii) a donor DNA template spanning a double stranded DNA break sitein the DNA junction polynucleotide, wherein the donor DNA templatecomprises a guide RNA hybridization site of the CgRRS, optionally a PAMsite of the CgRRS, and optionally DNA sequences of the DNA junctionpolynucleotide flanking the double stranded DNA break site; and(b) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant comprising the CgRRS.

50. The method of embodiment 47 or 48, wherein the CgRRS is introducedby:

(a) contacting the original transgenic locus with: (i) at least oneadenine base editor (ABE) and/or cytosine base pair editor (CBE); and(ii) a guide RNA comprising an RNA equivalent of the DNA locatedimmediately 5′ or 3′ to an original PAM site located within the DNAjunction polynucleotide of the original transgenic locus; and(b) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant comprising the CgRRS.

51. The method of embodiment 47 or 48, wherein the CgRRS is introducedby:

(a) contacting the original transgenic locus with: (i) a Zinc FingerNuclease or TALEN which recognizes a DNA junction polynucleotide of theoriginal transgenic locus or (ii) a Zinc Finger nickase or Tale nickasewhich recognizes a DNA junction polynucleotide of the originaltransgenic locus; and (iii) a donor DNA template spanning a doublestranded DNA break site in the DNA junction polynucleotide, wherein thedonor DNA template comprises a guide RNA hybridization site of theCgRRS, optionally a PAM site of the CgRRS, and optionally DNA sequencesof the DNA junction polynucleotide flanking the double stranded DNAbreak site; and(b) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant comprising the CgRRS.

52. The method of any one of embodiments 47 to 51, further comprisingcontacting the original transgenic locus with one or more gene editingmolecules that provide for excision or inactivation of a selectablemarker transgene of the original transgenic locus and selecting for atransgenic plant cell, transgenic plant part, or transgenic plantwherein the selectable marker transgene has been excised or inactivated.

53. The method of embodiment 52, wherein the gene editing moleculesinclude a donor DNA template or other DNA template containing anexpression cassette or coding region which confers a useful trait andthe transgenic plant cell, transgenic plant part, or transgenic plant isselected for integration of the expression cassette at the site of theselectable marker transgene excision or inactivation.

54. A method of excising a modified transgenic locus or portion thereoffrom an edited transgenic plant genome comprising the steps of:

(a) contacting the edited transgenic plant genome of any one ofembodiments 1 to 11 with: (i) an RNA dependent DNA endonuclease (RdDe);and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNAhybridization site of the first OgRRS and the first CgRRS; wherein theRdDe recognizes a OgRRS/gRNA and a CgRRS/gRNA hybridization complex;and,(b) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant wherein the modified transgenic locus or a portionthereof flanked by the first OgRRS and the first CgRRS has been excised.

55. The method of embodiment 54, wherein the edited transgenic plantgenome is contacted in step (a) by introducing one or more compositionscomprising or encoding the RdDe(s) and the gRNA into a transgenic plantcell comprising the edited transgenic plant genome.

56. The method of embodiment 54 or 55, wherein the edited transgenicplant genome is contacted in step (a) by introducing one or morecompositions comprising the gRNA into a transgenic plant cell comprisingthe edited transgenic plant genome, wherein the transgenic plant cellcomprises a polynucleotide that encodes the RdDe and the encoded RdDe isexpressed in the transgenic plant cell.

57. The method of any one of embodiments 54 to 56, wherein the editedtransgenic plant genome further comprises a second modified transgeniclocus that comprises a second OgRRS located in a first DNA junctionpolynucleotide of the second modified transgenic locus; and a secondCgRRS located in a second DNA junction polynucleotide of the secondmodified transgenic locus; and wherein the method further comprises:

(a) contacting the edited transgenic plant genome with: (i) an RNAdependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capableof hybridizing to the guide RNA hybridization site of the second OgRRSand the second CgRRS; wherein the RdDe recognizes a second OgRRS/gRNAand a second CgRRS/gRNA hybridization complex; and,(b) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant wherein the second modified transgenic locus or aportion thereof flanked by the second OgRRS and the second CgRRS hasbeen excised.

58. The method of any one of embodiments 54 to 57, wherein thetransgenic plant cell is in tissue culture, in a callus culture, a plantpart, or in a whole plant and/or wherein the transgenic plant cell is ahaploid plant cell.

59a. The method of any one of embodiments 54 to 58, wherein frequency ofexcision of the modified transgenic locus or a portion thereof in thetransgenic plant cells, transgenic plant parts, or transgenic plantssubjected to selection is greater than the frequency of excision of themodified transgenic locus or a portion thereof in control transgenicplant cells, transgenic plant parts, or transgenic plants treated withtwo control gRNAs and subjected to selection.

59b. The method of any one of embodiments 54 to 58, wherein the editedtransgenic plant genome is further contacted in step (a) with a donorDNA template, optionally wherein the donor DNA template comprises apolynucleotide sequence comprising non-transgenic plant genomic DNAlocated outside of the modified transgenic DNA which is to be excised,optionally wherein the donor DNA template comprises non-transgenic plantgenomic DNA present in an unmodified plant at the site where thetransgenic locus was originally inserted, and optionally in step (b)selecting a transgenic plant cell, transgenic plant part, or transgenicplant wherein polynucleotides in any of the donor DNA templates haveintegrated in the position in the plant genome where the modifiedtransgenic locus was excised.

60. A method of obtaining a plant breeding line comprising:

(a) crossing two transgenic plants comprising the edited transgenicgenomes of any of embodiments 1 to 11, wherein a first plant comprisingthe first modified transgenic locus is crossed to a second plantcomprising the second modified transgenic locus; and,(b) selecting a progeny plant comprising the first and second modifiedtransgenic locus from the cross, thereby obtaining a plant breedingline.

61. The method of embodiment 60, wherein the plant breeding line issubjected to a haploid inducer and a haploid plant breeding linecomprising at least the first and second breeding line is selected.

62. A method for obtaining inbred transgenic plant germplasm containingdifferent transgenic traits comprising:

(a) introgressing at least a first transgenic locus and a secondtransgenic locus into inbred germplasm to obtain a donor inbred parentplant line comprising the first and second transgenic loci, wherein afirst OgRRS and a first CgRRS are operably linked to both DNA junctionpolynucleotides of at least the first transgenic locus and optionallywherein a second OgRRS and a second CgRRS are operably linked to thesecond transgenic locus;(b) contacting the transgenic plant genome of the donor inbred parentplant line with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii)a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridizationsite of the first OgRRS and the second CgRRS; wherein the RdDerecognizes a first OgRRS/gRNA and a first CgRRS/gRNA hybridizationcomplex; and(c) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant comprising an edited transgenic plant genome in theinbred germplasm, wherein the first transgenic locus has been excisedand the second transgenic locus is present in the inbred germplasm.

63. The method of embodiment 62, wherein the introgression comprisescrossing germplasm comprising the first and/or second transgenic plantlocus with the inbred germplasm, selecting progeny comprising the firstor second transgenic plant locus, and crossing the selected progeny withthe inbred germplasm as a recurrent parent.

64. The method of embodiment 62 or 63, further comprising contacting thetransgenic plant genome in step (b) with one or more gene editingmolecules that provide for excision or inactivation of a selectablemarker transgene of the second transgenic locus and selecting for atransgenic plant cell, transgenic plant part, or transgenic plantwherein the selectable marker transgene has been excised or inactivated.

65. The method of any one of embodiments 62 to 64, wherein the geneediting molecules include a donor DNA template containing an expressioncassette or coding region which confers a useful trait and thetransgenic plant cell, transgenic plant part, or transgenic plant isselected for integration of the expression cassette at the site of theselectable marker transgene excision or inactivation.

66. The method of any one of embodiments 62 to 65, wherein a secondOgRRS and a second CgRRS are operably linked to the second transgeniclocus and wherein the method further comprises contacting the transgenicplant genome of the donor inbred parent plant line with: (i) an RNAdependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capableof hybridizing to the guide RNA hybridization site of the second OgRRSand the second CgRRS; wherein the RdDe recognizes a second OgRRS/gRNAand a second CgRRS/gRNA hybridization complex in step (b); and selectinga transgenic plant cell, transgenic plant part, or transgenic plantwherein the second transgenic locus has been excised in step (c).

67. The method of any one of embodiments 62 to 66, wherein thetransgenic plant genome is contacted in step (b) by introducing one ormore compositions comprising or encoding the RdDe(s) and gRNAs into atransgenic plant cell comprising the transgenic plant genome.

68. The method of any one of embodiments 62 to 66, wherein the editedtransgenic plant genome is contacted in step (a) by introducing one ormore compositions comprising the gRNA into a transgenic plant cellcomprising the edited transgenic plant genome, wherein the transgenicplant cell comprises a polynucleotide that encodes the RdDe and theencoded RdDe is expressed in the transgenic plant cell.

69. The method of any one of embodiments 62 to 68, wherein thetransgenic plant genome is further contacted in step (b) with a donorDNA template molecule comprising an introduced transgene and atransgenic plant cell comprising an edited transgenic plant genomecomprising an insertion of the introduced transgene in the firsttransgenic locus is selected in step (c).

70. The method of any one of embodiments 62 to 69, wherein thetransgenic plant genome is further contacted in step (b) with: (i) adonor DNA template molecule comprising an introduced transgene; and (ii)one or more DNA editing molecules which introduce a double stranded DNAbreak in the second transgenic locus; and a transgenic plant cellcomprising an edited transgenic plant genome comprising an insertion ofthe introduced transgene in the second transgenic locus is selected instep (b).

71. The method of any one of embodiments 62 to 70, further comprising:

(d) contacting the edited transgenic plant genome in the selectedtransgenic plant cell of step (c) with: (i) a donor DNA templatemolecule comprising an introduced transgene; and (ii) one or more DNAediting molecules which introduce a double stranded DNA break in or nearthe excision site of the first transgenic locus or in the secondtransgenic locus; and,(e) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant comprising a further edited transgenic plant genomecomprising an insertion of the introduced transgene in or near theexcision site of the first transgenic locus or in the second transgeniclocus.

72. The method of any one of embodiments 62 to 71, wherein thetransgenic plant germplasm is transgenic corn plant germplasm andwherein the first and/or second modified transgenic locus comprises atleast one modification of a Bt11, DAS-59122-7, DP-4114, GA21, MON810,MON87411, MON87427, MON88017, MON89034, MIR162, MIR604, NK603,SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038,MON863, MON87403, MON87403, MON87419, MON87460, MZHG0JG, MZIR098,VCO-Ø1981-5, 98140, or TC1507 original transgenic locus in a transgeniccorn plant genome, wherein the modification comprises the CgRRS in thesecond DNA junction polynucleotide of the first transgenic locus andoptionally the CgRRS in the second DNA junction polynucleotide of thetransgenic locus, and wherein the modification optionally furthercomprises a deletion of at least one selectable marker gene and/ornon-essential DNA in the original transgenic locus.

73. The method of any one of embodiments 62 to 72, wherein thetransgenic plant germplasm is transgenic soybean plant germplasm andwherein the first and/or second modified transgenic locus comprises amodification of an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 originaltransgenic locus in a transgenic soybean plant genome, wherein themodification comprises the CgRRS in the second DNA junctionpolynucleotide of the first transgenic locus and optionally the CgRRS inthe second DNA junction polynucleotide of the transgenic locus, andwherein the modification optionally further comprises a deletion of atleast one selectable marker gene and/or non-essential DNA in theoriginal transgenic locus.

74. The method of any one of embodiments 62 to 72, wherein thetransgenic plant germplasm is transgenic cotton plant germplasm andwherein the first, second, and/or third modified transgenic locuscomprises at least one modification of a DAS-21023-5, DAS-24236-5,COT102, LLcotton25, MON15985, MON88701, or MON88913 original transgeniclocus in a transgenic cotton plant genome, wherein the modificationcomprises the CgRRS in the second DNA junction polynucleotide of thefirst transgenic locus and optionally the CgRRS in the second DNAjunction polynucleotide of the transgenic locus, and wherein themodification optionally further comprises a deletion of at least oneselectable marker gene and/or non-essential DNA in the originaltransgenic locus.

75. The method of any one of embodiments 62 to 72, wherein thetransgenic plant germplasm is transgenic canola plant germplasm andwherein the first, second, and or third modified transgenic locuscomprises a modification of an GT73, HCN28, MON88302, or MS8 originaltransgenic locus in a transgenic canola plant genome, wherein themodification comprises the CgRRS in the second DNA junctionpolynucleotide of the first transgenic locus and optionally the CgRRS inthe second DNA junction polynucleotide of the transgenic locus, andwherein the modification optionally further comprises a deletion of atleast one selectable marker gene and/or non-essential DNA in theoriginal transgenic locus.

EXAMPLES Example 1. Introduction of CgRRS in 5′ or 3′ JunctionPolynucleotides of Transgenic Loci

Transgenic plant genomes containing one or more of the followingtransgenic loci (events) are contacted with:

(i) an ABE or CBE and guide RNAs which recognize the indicated targetDNA sites (guide RNA coding plus PAM site) in the 5′ or 3′ junctionpolynucleotides of the event to introduce a CgRRS in the junctionpolynucleotide; or(ii) an RdDe and guide RNAs which recognize the indicated target DNAsite (guide RNA coding plus PAM site) in the 5′ or 3′ junctionpolynucleotides of the event as well as a donor DNA template spanningthe double stranded DNA break site in the junction polynucleotide tointroduce a CgRRS in a junction polynucleotide. Plant cells, callus,parts, or whole plants comprising the introduced CgRRS in the transgenicplant genome are selected.

TABLE 6 Examples of OgRRS in Corn Events CORN EVENT NAME OgRRS ExamplesCgRRS MIR162 tttatagatcatacaaaaaggcccagt Introduced into the 5′(SEQ ID NO: 35; in 3′ junction polynucleotide junction polynucleotide ofof SEQ ID NO: 5 SEQ ID NO: 5) tttaatgtactgaattgtctagaccc(SEQ ID NO: 44; in 3′ junction polynucleotide of SEQ ID NO: 5)tttaatgtactgaattgtctagacccg (SEQ ID NO: 45; in 3′junction polynucleotide of SEQ ID NO: 5) DP-4114tttgtagcacttgcacgtagttacccg Introduced into the 3′ (SEQ ID NO: 48; in 5′junction polynucleotide junction polynucleotide of of SEQ ID NO: 2SEQ ID NO: 2)

Example 2. Use of an RdDe, Guide RNA, and a Donor DNA Template Insertionto Introduce a CgRRS in a MIR162 Junction Polynucleotide

Two plant gene expression vectors are prepared. Plant expressioncassettes for expressing a bacteriophage lambda exonuclease, abacteriophage lambda beta SSAP protein, and an E. coli SSB areconstructed essentially as set forth in US Patent ApplicationPublication 20200407754, which is incorporated herein by reference inits entirety. A DNA sequence encoding a tobacco c2 nuclear localizationsignal (NLS) is fused in-frame to the DNA sequences encoding theexonuclease, the bacteriophage lambda beta SSAP protein, and the E. coliSSB to provide a DNA sequence encoding the c2 NLS-Exo, c2 NLS lambdabeta SSAP, and c2 NLS-SSB fusion proteins that are set forth in SEQ IDNO: 135, SEQ ID NO: 134, and SEQ ID NO: 133 of US Patent ApplicationPublication 20200407754, respectively, and incorporated herein byexample. DNA sequences encoding the c2 NLS-Exo, c2 NLS lambda beta SSAP,and c2NLS-SSB fusion proteins are operably linked to a OsUBI1, ZmUBI1,OsACT promoter and a OsUbi1, ZmUBI1, OsACT polyadenylation siterespectively, to provide the exonuclease, SSAP, and SSB plant expressioncassettes.

A donor DNA template sequence (SEQ ID NO: 37) that targets the 5′ DNAjunction polynucleotide of the MIR162 event for insertion of a 27 basepair heterologous sequence, that is identical to a Cas12a recognitionsite at the 3′-junction of the MIR162 T-DNA insert, by HDR isconstructed. The donor DNA template sequence includes a replacementtemplate with desired insertion region (27 base pair long sequence ofSEQ ID NO: 35) flanked on both sides by homology arms of about 500-635bp in length. The homology arms match (i.e., are homologous to) gDNA(genomic DNA) regions flanking the target gDNA insertion site. Thereplacement template region comprising the donor DNA template is flankedat each end by DNA sequences identical to the MIR162 5′ polynucleotidesequence recognized by an RNA-guided nuclease and one or more gRNA(s)(e.g. gRNAs encoded by SEQ ID NO: 38, 39, and 40). In certain cases, adouble-stranded is made in the targeted MIR162 5′ polynucleotidesequence (e.g., using a Cas12a endonuclease and a gRNA encoded by SEQ IDNO: 38, 39, or 40) to allow for HDR with the donor DNA template andintroduction of the CgRRS. In certain cases, a deletion is made in thetargeted MIR162 5′ polynucleotide sequence (e.g., using a Cas12aendonuclease and gRNAs encoded by SEQ ID NO: 38 and 39 in combination orby using gRNAs encoded by SEQ ID NO: 39 and 40 in combination) to allowfor HDR with the donor DNA template and introduction of the CgRRS.

A plant expression cassette that provides for expression of theRNA-guided sequence-specific Cas12a endonuclease is constructed. A plantexpression cassette that provides for expression of a guide RNAcomplementary to sequences adjacent to the insertion site (e.g. gRNAsencoded by SEQ ID NO: 38, 39, and 40) are constructed. An Agrobacteriumsuperbinary plasmid transformation vector containing a cassette thatprovides for the expression of the phosphinothricin N-acetyltransferasesynthase (PAT) protein is constructed. Once the cassettes, donorsequence and Agrobacterium superbinary plasmid transformation vector areconstructed, they were combined to generate two maize transformationplasmids.

A maize transformation plasmid is constructed with the PAT cassette, theRNA-guided sequence-specific endonuclease cassette, the guide RNAcassette, and the MIR162 5′-junction polynucleotide donor DNA templatesequence into the Agrobacterium superbinary plasmid transformationvector (the control vector).

A maize transformation plasmid is constructed with the PAT cassette, theRNA-guided sequence-specific endonuclease cassette, the guide RNAcassette, the SSB cassette, the lambda beta SSAP cassette, the Exocassette, and the MIR162 5′-junction polynucleotide donor DNA templateinto the Agrobacterium superbinary plasmid transformation vector (thelambda red vector).

All constructs are transformed into Agrobacterium strain LBA4404.

Maize transformations are performed based on published methods (Ishidaet. al, Nature Protocols 2007; 2, 1614-1621). Briefly, immature embryosfrom inbred line GIBE0104, approximately 1.8-2.2 mm in size, areisolated from surface sterilized ears 10-14 days after pollination.Embryos are placed in an Agrobacterium suspension made with infectionmedium at a concentration of OD 600=1.0. Acetosyringone (200 μM) isadded to the infection medium at the time of use. Embryos andAgrobacterium are placed on a rocker shaker at slow speed for 15minutes. Embryos are then poured onto the surface of a plate ofco-culture medium. Excess liquid media is removed by tilting the plateand drawing off all liquid with a pipette. Embryos are flipped asnecessary to maintain a scutelum up orientation. Co-culture plates areplaced in a box with a lid and cultured in the dark at 22° C. for 3days. Embryos are then transferred to resting medium, maintaining thescutellum up orientation. Embryos remain on resting medium for 7 days at27-28° C. Embryos that produced callus are transferred to Selection 1medium with 7.5 mg/L phosphinothricin (PPT) and cultured for anadditional 7 days. Callused embryos are placed on Selection 2 mediumwith 10 mg/L PPT and cultured for 14 days at 27-28° C. Growing calliresistant to the selection agent are transferred to Pre-Regenerationmedia with 10 mg/L PPT to initiate shoot development. Calli remained onPre-Regeneration media for 7 days. Calli beginning to initiate shootsare transferred to Regeneration medium with 7.5 mg/L PPT in Phytatraysand cultured in light at 27-28° C. Shoots that reached the top of thePhytatray with intact roots are isolated into Shoot Elongation mediumprior to transplant into soil and gradual acclimatization to greenhouseconditions.

When a sufficient amount of viable tissue is obtained, it can bescreened for insertion at the MIR162 junction sequence, using aPCR-based approach. The PCR primer on the 5′-end can be5′-ttttatgtattatttggtccctaca-3′ (SEQ ID NO: 41) and the. PCR primer onthe 3′-end is 5′-gtcgacggcgtttaacaggctggca-3′ (SEQ ID NO: 42). Theseprimers that flank donor DNA homology arms are used to amplify theMIR162 5′-junction sequence. The correct donor sequence insertion willproduce a 1579 bp product. Amplicons can be sequenced directly using anamplicon sequencing approach or ligated to a convenient plasmid vectorfor Sanger sequencing. The CgRRS which is introduced into a 5′ junctionpolynucleotide of MIR162 comprises SEQ ID NO: 43. Those plants in whichthe MIR162 5′ junction polynucleotide sequence now contains the intendedCgRRS (i.e. Cas12a recognition sequence) are selected and grown tomaturity. The T-DNA encoding the Cas12a reagents can be segregated awayfrom the modified junction sequence in a subsequent generation. Theresultant INIR12 transgenic locus comprising the CgRRS and OgRRS (e.g.which each comprise SEQ ID NO: 35 and an operably linked PAM site) canbe excised using Cas12a and a suitable gRNA which hybridizes to DNAcomprising SEQ ID NO: 35 at both the OgRRS and the CgRRS.

Example 3. Insertion of a CgRRS Element in the 3′-Junction of theDP-4114 Event

Two plant gene expression vectors are prepared. Plant expressioncassettes for expressing a bacteriophage lambda exonuclease, abacteriophage lambda beta SSAP protein, and an E. coli SSB areconstructed essentially as set forth in US Patent ApplicationPublication 20200407754, which is incorporated herein by reference inits entirety and as described in Example 2.

A DNA donor sequence (SEQ ID NO: 47) that targets the 3′-T-DNA junctionpolynucleotide of the DP-4114 event (SEQ ID NO:1; FIG. 2 ) forHDR-mediated insertion of a 27 base pair OgRRS sequence (SEQ ID NO: 48)that is identical to a Cas12a recognition site at the 5′-junctionpolynucleotide of the DP-4114 T-DNA insert is constructed. The DNA donorsequence includes a replacement template with desired insertion region(27 base pairs long) flanked on both sides by homology arms about500-635 bp in length. The homology arms match (i.e., are homologous to)gDNA (genomic DNA) regions flanking the target gDNA insertion site. Thereplacement template region comprising the donor DNA is flanked at eachend by DNA sequences identical to the DP-4114 3′-T-DNA junctionpolynucleotide sequence recognized by an RNA-guided nuclease and a gRNA(e.g., encoded by SEQ ID NO: 49).

A plant expression cassette that provides for expression of theRNA-guided sequence-specific Cas12a endonuclease is constructed. A plantexpression cassette that provides for expression of a guide RNA (e.g.,encoded by SEQ ID NO: 49) complementary to sequences adjacent to theinsertion site is constructed. An Agrobacterium superbinary plasmidtransformation vector containing a cassette that provides for theexpression of the phosphinothricin N-acetyltransferasesynthase (PAT)protein is constructed. Once the cassettes, donor sequence andAgrobacterium superbinary plasmid transformation vector are constructed,they are combined to generate two maize transformation plasmids.

A maize transformation plasmid is constructed with the PAT cassette, theRNA-guided sequence-specific endonuclease cassette, the guide RNAcassette, and the DP-4114 3′-T_DNA junction sequence DNA donor sequenceinto the Agrobacterium superbinary plasmid transformation vector (thecontrol vector).

A maize transformation plasmid is constructed with the PAT cassette, theRNA-guided sequence-specific endonuclease cassette, the guide RNAcassette, the SSB cassette, the lambda beta SSAP cassette, the Exocassette, and the DP-4114 3′-T_DNA junction sequence DNA donor sequence(SEQ ID NO: 47) into the Agrobacterium superbinary plasmidtransformation vector (the lambda red vector).

All constructs are transformed into Agrobacterium strain LBA4404.

Maize transformations are performed based on published methods (Ishidaet. al, Nature Protocols 2007; 2, 1614-1621). Briefly, immature embryosfrom inbred line GIBE0104, approximately 1.8-2.2 mm in size, areisolated from surface sterilized ears 10-14 days after pollination.Embryos are placed in an Agrobacterium suspension made with infectionmedium at a concentration of OD 600=1.0. Acetosyringone (200 μM) isadded to the infection medium at the time of use. Embryos andAgrobacterium are placed on a rocker shaker at slow speed for 15minutes. Embryos are then poured onto the surface of a plate ofco-culture medium. Excess liquid media is removed by tilting the plateand drawing off all liquid with a pipette. Embryos are flipped asnecessary to maintain a scutelum up orientation. Co-culture plates areplaced in a box with a lid and cultured in the dark at 22° C. for 3days. Embryos are then transferred to resting medium, maintaining thescutellum up orientation. Embryos remain on resting medium for 7 days at27-28° C. Embryos that produced callus are transferred to Selection 1medium with 7.5 mg/L phosphinothricin (PPT) and cultured for anadditional 7 days. Callused embryos are placed on Selection 2 mediumwith 10 mg/L PPT and cultured for 14 days at 27-28° C. Growing calliresistant to the selection agent are transferred to Pre-Regenerationmedia with 10 mg/L PPT to initiate shoot development. Calli remained onPre-Regeneration media for 7 days. Calli beginning to initiate shootsare transferred to Regeneration medium with 7.5 mg/L PPT in Phytatraysand cultured in light at 27-28° C. Shoots that reached the top of thePhytatray with intact roots are isolated into Shoot Elongation mediumprior to transplant into soil and gradual acclimatization to greenhouseconditions.

When a sufficient amount of viable tissue is obtained, it can bescreened for insertion at the DP-4114 junction sequence, using aPCR-based approach. The PCR primer on the 5′-end is5′-tacgctgggccctggaaggctagga-3′ (SEQ ID NO: 50). The PCR primer on the3′-end is 5′-gatggacgagacgaggcggtggaga-3′ (SEQ ID NO: 51). The aboveprimers that flank donor DNA homology arms are used to amplify theDP-4114 3′-junction polynucleotide sequence. The correct donor sequenceinsertion will produce a 1563 bp product. A unique DNA fragmentcomprising the CgRRS in the DP-4114 3′ junction polynucleotide is setforth in SEQ ID NO: 52. Amplicons can be sequenced directly using anamplicon sequencing approach or ligated to a convenient plasmid vectorfor Sanger sequencing. Those plants in which the DP-4114 junctionsequence now contains the intended Cas12a recognition sequence areselected and grown to maturity. The T-DNA encoding the Cas12a reagentscan be segregated away from the modified junction sequence in asubsequent generation. The resultant INIR6 transgenic locus (SEQ ID NO:53) comprising the CgRRS and OgRRS (e.g. which each comprise SEQ ID NO:48) can be excised using Cas12a and a suitable gRNA which hybridizes toDNA comprising SEQ ID NO: 48 at both the OgRRS and the CgRRS.

The breadth and scope of the present disclosure should not be limited byany of the above-described embodiments.

What is claimed is:
 1. An edited transgenic plant genome comprising afirst modified transgenic locus that comprises: (i) a first originatorguide RNA recognition site (OgRRS) comprising a protospacer adjacentmotif (PAM) site operably linked to a guide RNA hybridization site,wherein the OgRRS is located in transgenic DNA, in non-transgenic plantgenomic DNA, or in a combination thereof in a first DNA junctionpolynucleotide of the first modified transgenic locus; and (ii) a firstcognate guide RNA recognition site (CgRRS) comprising a protospaceradjacent motif (PAM) site operably linked to a guide RNA hybridizationsite located in a second DNA junction polynucleotide of the firstmodified transgenic locus, wherein the CgRRS is absent from transgenicplant genomes comprising a first original transgenic locus that isunmodified and wherein the OgRRS and the CgRRS can hybridize to onefirst guide RNA (gRNA).
 2. The edited transgenic plant genome of claim1, wherein the guide RNA hybridization site of the OgRRS and the CgRRScomprise at least 18 nucleotides of identical DNA sequence.
 3. Theedited transgenic plant genome of claim 1, wherein the OgRRS and theCgRRS can be cleaved by the same RNA dependent DNA endonuclease (RdDe)when the OgRRS and the CgRRS are hybridized to the one first gRNA. 4.The edited transgenic plant genome of claim 3, wherein the RdDe is aclass 2 type II or class 2 type V RdDe.
 5. The edited transgenic plantgenome of claim 1, wherein the PAM site in the OgRRS and the PAM site inthe CgRRS comprise distinct PAM sequences that are recognized by thesame RdDe when the OgRRS and the CgRRS are hybridized to the one gRNA.6. The edited transgenic plant genome of claim 1, wherein the OgRRS islocated in non-transgenic DNA or in non-transgenic DNA and within about1000, 750, 500, 250, 100, or 50 base pairs (bp) of transgenic DNA of thefirst DNA junction polynucleotide and/or wherein the CgRRS is located innon-transgenic DNA or in non-transgenic DNA and within about 1000, 750,500, 250, 100, or 50 bp of transgenic DNA in the second DNA junctionpolynucleotide.
 7. The edited transgenic plant genome of claim 1,wherein the edited transgenic plant genome further comprises a secondmodified transgenic locus that comprises: (i) a second OgRRS located intransgenic DNA, non-transgenic plant genomic DNA, or in a combinationthereof in a first DNA junction polynucleotide of the second modifiedtransgenic locus; and (ii) a second CgRRS located in a second DNAjunction polynucleotide of the second modified transgenic locus, whereinthe CgRRS is absent from unedited transgenic plant genomes comprising asecond original transgenic locus, optionally wherein the second OgRRSand the second CgRRS can hybridize to one second guide RNA (gRNA) andoptionally wherein the second guide RNA is not complementary to thefirst OgRRS and the first CgRRS.
 8. The edited transgenic plant genomeof claim 1, wherein the first modified transgenic locus comprises atleast one modification of a Bt11, DAS-59122-7, DP-4114, GA21, MON810,MON87411, MON87427, MON88017, MON89034, MIR162, MIR604, NK603,SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038,MON863, MON87403, MON87403, MON87419, MON87460, MZHG0JG, MZIR098,VCO-Ø1981-5, 98140, or TC1507 original transgenic locus in a transgeniccorn plant genome, wherein the modification comprises the CgRRS in thesecond DNA junction polynucleotide of the first transgenic locus, andoptionally wherein the modification further comprises a deletion of atleast one selectable marker gene and/or non-essential DNA in theoriginal transgenic locus.
 9. The edited transgenic plant genome ofclaim 1, wherein the first modified transgenic locus comprises amodification of an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 originaltransgenic locus in a transgenic soybean plant genome, wherein themodification comprises the CgRRS in the second DNA junctionpolynucleotide of the first transgenic locus, and wherein themodification optionally further comprises a deletion of at least oneselectable marker gene and/or non-essential DNA in the originaltransgenic locus.
 10. The edited transgenic plant genome of claim 1,wherein the first modified transgenic locus comprises at least onemodification of a DAS-21023-5, DAS-24236-5, COT102, LLcotton25,MON15985, MON88701, or MON88913 original transgenic locus in atransgenic cotton plant genome, wherein the modification comprises theCgRRS in the second DNA junction polynucleotide of the first transgeniclocus and optionally the CgRRS in the second DNA junction polynucleotideof the second modified transgenic locus, and wherein the modificationoptionally further comprises a deletion of at least one selectablemarker gene and/or non-essential DNA in the original transgenic locus.11. The edited transgenic plant genome of claim 1, wherein the firstmodified transgenic locus comprises a modification of an GT73, HCN28,MON88302, or MS8 original transgenic locus in a transgenic canola plantgenome, wherein the modification comprises the CgRRS in the second DNAjunction polynucleotide of the first transgenic locus, and wherein themodification optionally further comprises a deletion of at least oneselectable marker gene and/or non-essential DNA in the originaltransgenic locus.
 12. The edited transgenic plant genome of any one ofclaims to 1 to 11, wherein the first and/or second modified transgeniclocus lacks a selectable marker transgene which confers resistance to anantibiotic, tolerance to an herbicide, or an ability to grow on aspecific carbon source, optionally wherein the selectable markertransgene was present in the original transgenic locus and/or whereinthe specific carbon source is optionally mannose.
 13. The editedtransgenic plant genome of any one of claims 1 to 11, wherein: (i) thefirst CgRRS is located in non-transgenic plant genomic DNA of the secondDNA junction polynucleotide of the first modified transgenic locus; or(ii) the first CgRRS is located in transgenic DNA of the second DNAjunction polynucleotide of the first modified transgenic locus.
 14. Theedited transgenic plant genome of claim 13, wherein the CgRRS is locatedin the non-transgenic plant genomic DNA and comprises a sequence having50% to 70% sequence identity to the non-transgenic plant genomic DNAlocated at the same chromosomal location in the original transgeniclocus.
 15. The edited transgenic plant genome of claims 1 to 11, whereinthe first and/or second transgenic locus further comprise a secondintroduced transgene or optionally wherein the second introducedtransgene is integrated at a site in the modified transgenic locus whichwas occupied by a selectable marker transgene in the original transgeniclocus.
 16. The edited transgenic plant genome of any one of claims 1 to11, wherein the genome further comprises a targeted genetic change. 17.A transgenic plant cell comprising the edited transgenic plant genome ofany one of claims 1 to
 11. 18. A transgenic plant comprising thetransgenic plant genome of any one of claims 1 to
 11. 19. A transgenicplant part comprising the edited transgenic plant genome of any one ofclaims 1 to
 11. 20. The transgenic plant part of claim 19, wherein thepart is a seed, leaf, tuber, stem, root, or boll.
 21. A method forobtaining a bulked population of inbred seed for commercial seedproduction comprising selfing the transgenic plant of claim 18 andharvesting seed from the selfed crop plants.
 22. A method of obtaininghybrid crop seed comprising crossing a first crop plant comprising thetransgenic plant of claim 18 to a second crop plant and harvesting seedfrom the cross.
 23. The method of claim 22, wherein the first crop plantand the second crop plant are in distinct heterotic groups.
 24. Themethod of claim 22, wherein either the first or second crop plant arepollen recipients which have been rendered male sterile.
 25. The methodof claim 24, wherein the crop plant is rendered male sterile byemasculation, cytoplasmic male sterility, a chemical hybridizing agentor system, a transgene, and/or a mutation in an endogenous plant gene.26. The method of any one of claims 22 to 25, further comprising thestep of sowing the hybrid crop seed.
 27. A DNA molecule comprising thecognate guide RNA recognition site (CgRRS) and at least 10 bp oftransgenic DNA or non-transgenic plant genomic DNA flanking the CgRRS,wherein the transgenic DNA or non-transgenic plant genomic DNA comprisesDNA sequences of the second DNA junction polynucleotide of the firstmodified transgenic locus set forth in any one of claims 1 to
 11. 28.The DNA of claim 27, wherein the modified transgenic locus is a Bt11,DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017,MON89034, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138,DP-33121, HCEM485, LY038, MON863, MON87403, MON87403, MON87419,MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, or TC1507 transgeniclocus.
 29. The DNA of claim 27, wherein the modified transgenic locus isan A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701,MON87708, MON89788, MST-FGØ72-3, and/or SYHT0H2 transgenic locus. 30.The DNA of claim 27 wherein the modified transgenic locus is: (i) aDAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/orMON88913 transgenic locus and wherein the modifications optionallyfurther comprise a deletion of at least one selectable marker geneand/or non-essential DNA in the original transgenic locus; or (ii)wherein the modified transgenic locus is a GT73, HCN28, MON88302, or MS8transgenic locus.
 31. The DNA of any one of claims 27 to 30, wherein theDNA is purified or isolated.
 32. A processed transgenic plant productcontaining the DNA of any one of claims 27 to
 30. 33. A biologicalsample containing the DNA of any one of claims 27 to
 30. 34. A nucleicacid marker adapted for detection of genomic DNA or fragments thereofcomprising a cognate guide RNA recognition site (CgRRS) in, adjacent to,or operably linked to a DNA junction polynucleotide of a modifiedtransgenic locus.
 35. The nucleic acid marker of claim 34, comprising apolynucleotide of at least 18 nucleotides in length which spans DNAsequences comprising both the CgRRS and non-transgenic plant genomic DNAflanking either a telomere-proximal and a centromere proximal end of theCgRRS.
 36. The nucleic acid marker of claim 34, wherein the markerfurther comprises a detectable label.
 37. The nucleic acid marker ofclaim 34, wherein the CgRRS and non-transgenic plant genomic DNA areidentical to sequences in a modified transgenic locus.
 38. The nucleicacid marker of claim 34, wherein the modified transgenic locus is aBt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017,MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121,HCEM485, LY038, MON863, MON87403, MON87403, MON87419, MON87460, MZHG0JG,MZIR098, VCO-Ø1981-5, 98140, or TC1507 transgenic locus comprising theCgRRS, wherein the CgRRS is located in, adjacent to, or operably linkedto a DNA junction polynucleotide of the modified transgenic locus. 39.The nucleic acid marker of claim 34, wherein the modified transgeniclocus is a modified A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 transgeniclocus comprising the CgRRS, wherein the CgRRS is located in, adjacentto, or operably linked to a DNA junction polynucleotide of the modifiedtransgenic locus.
 40. The nucleic acid marker of claim 34, wherein themodified transgenic locus is a DAS-21023-5, DAS-24236-5, COT102,LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locuscomprising the CgRRS, wherein the CgRRS is located in, adjacent to, oroperably linked to a DNA junction polynucleotide of the modifiedtransgenic locus.
 41. The nucleic acid marker of claim 34, wherein themodified transgenic locus is a GT73, HCN28, MON88302, or MS8 transgeniclocus comprising the CgRRS, wherein the CgRRS is located in, adjacentto, or operably linked to a DNA junction polynucleotide of the modifiedtransgenic locus.
 42. A processed transgenic plant product obtained fromthe transgenic plant part of claim 19 or 20, wherein the processed plantproduct contains a polynucleotide comprising a cognate guide RNArecognition site (CgRRS) and at least 10 bp of transgenic DNA ornon-transgenic plant genomic DNA flanking the CgRRS, wherein thetransgenic DNA or non-transgenic plant genomic DNA comprises transgenicor non-transgenic plant genomic DNA sequences of the second DNA junctionpolynucleotide of the first modified transgenic locus.
 43. A biologicalsample obtained from the transgenic plant cell of claim 17, thetransgenic plant of claim 18, or the transgenic plant part of claim 19or 20, wherein the biological sample contains one or morepolynucleotide(s) comprising the a cognate guide RNA recognition site(CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plantgenomic DNA flanking the CgRRS, wherein the transgenic DNA ornon-transgenic plant genomic DNA comprises transgenic or non-transgenicplant genomic DNA sequences of the second DNA junction polynucleotide ofthe first modified transgenic locus.
 44. A method of detecting theedited transgenic plant genome of any one of claims 1 to 11, comprisingthe step of detecting the presence of a polynucleotide comprising one ormore of said CgRRS.
 45. The method of claim 44, wherein thepolynucleotide is detected by detecting a single nucleotide polymorphism(SNP) in the CgRRS that is present in the modified transgenic locus butabsent in the original transgenic locus.
 46. The method of claim 44,wherein the edited transgenic plant genome is detected in a transgenicplant cell, a transgenic plant part, a transgenic plant, a processedtransgenic plant product, or a biological sample.
 47. A method ofobtaining an edited transgenic plant genome comprising a modifiedtransgenic locus comprising the step of introducing a cognate guide RNArecognition site (CgRRS) in a DNA junction polynucleotide of an originaltransgenic locus, wherein the CgRRS is in, adjacent to, or operablylinked to a DNA junction polynucleotide of the modified transgeniclocus.
 48. The method of claim 47, wherein the CgRRS is located innon-transgenic plant genomic DNA of the DNA junction polynucleotide ofthe modified transgenic locus and optionally wherein the CgRRS comprisesa sequence having 50% to 70% sequence identity to the non-transgenicplant genomic DNA located at the same chromosomal location in theoriginal transgenic locus.
 49. The method of claim 47, wherein the CgRRSis introduced by: (a) contacting the original transgenic locus with: (i)an RNA dependent DNA endonuclease (RdDe) or RdDe nickase; and a guideRNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′to an original PAM site located within the DNA junction polynucleotideof the original transgenic locus; or (ii) a guide RNA comprising an RNAequivalent of the DNA located immediately 5′ or 3′ to an original PAMsite located within the DNA junction polynucleotide of the originaltransgenic locus; and (iii) a donor DNA template spanning a doublestranded DNA break site in the DNA junction polynucleotide, wherein thedonor DNA template comprises a guide RNA hybridization site of theCgRRS, optionally a PAM site of the CgRRS, and optionally DNA sequencesof the DNA junction polynucleotide flanking the double stranded DNAbreak site; and (b) selecting a transgenic plant cell, transgenic plantpart, or transgenic plant comprising the CgRRS.
 50. The method of claim47, wherein the CgRRS is introduced by: (a) contacting the originaltransgenic locus with: (i) at least one adenine base editor (ABE) and/orcytosine base pair editor (CBE); and (ii) a guide RNA comprising an RNAequivalent of the DNA located immediately 5′ or 3′ to an original PAMsite located within the DNA junction polynucleotide of the originaltransgenic locus; and (b) selecting a transgenic plant cell, transgenicplant part, or transgenic plant comprising the CgRRS.
 51. The method ofclaim 47, wherein the CgRRS is introduced by: (a) contacting theoriginal transgenic locus with: (i) a Zinc Finger Nuclease or TALENwhich recognizes a DNA junction polynucleotide of the originaltransgenic locus or (ii) a Zinc Finger nickase or Tale nickase whichrecognizes a DNA junction polynucleotide of the original transgeniclocus; and (iii) a donor DNA template spanning a double stranded DNAbreak site in the DNA junction polynucleotide, wherein the donor DNAtemplate comprises a guide RNA hybridization site of the CgRRS,optionally a PAM site of the CgRRS, and optionally DNA sequences of theDNA junction polynucleotide flanking the double stranded DNA break site;and (b) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant comprising the CgRRS.
 52. The method of claim 47,further comprising contacting the original transgenic locus with one ormore gene editing molecules that provide for excision or inactivation ofa selectable marker transgene of the original transgenic locus andselecting for a transgenic plant cell, transgenic plant part, ortransgenic plant wherein the selectable marker transgene has beenexcised or inactivated.
 53. The method of claim 52, wherein the geneediting molecules include a donor DNA template or other DNA templatecontaining an expression cassette or coding region which confers auseful trait and the transgenic plant cell, transgenic plant part, ortransgenic plant is selected for integration of the expression cassetteat the site of the selectable marker transgene excision or inactivation.54. A method of excising a modified transgenic locus or portion thereoffrom an edited transgenic plant genome comprising the steps of: (a)contacting the edited transgenic plant genome of any one of claims 1 to11 with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guideRNA (gRNA) capable of hybridizing to the guide RNA hybridization site ofthe first OgRRS and the first CgRRS; wherein the RdDe recognizes aOgRRS/gRNA and a CgRRS/gRNA hybridization complex; and, (b) selecting atransgenic plant cell, transgenic plant part, or transgenic plantwherein the modified transgenic locus or portion thereof flanked by thefirst OgRRS and the first CgRRS has been excised.
 55. The method ofclaim 54, wherein the edited transgenic plant genome is contacted instep (a) by introducing one or more compositions comprising or encodingthe RdDe(s) and the gRNA into a transgenic plant cell comprising theedited transgenic plant genome.
 56. The method of claim 54, wherein theedited transgenic plant genome is contacted in step (a) by introducingone or more compositions comprising the gRNA into a transgenic plantcell comprising the edited transgenic plant genome, wherein thetransgenic plant cell comprises a polynucleotide that encodes the RdDeand the encoded RdDe is expressed in the transgenic plant cell.
 57. Themethod of claim 54, wherein the edited transgenic plant genome furthercomprises a second modified transgenic locus that comprises a secondOgRRS located in non-transgenic plant genomic DNA of a first DNAjunction polynucleotide of the second modified transgenic locus; and asecond CgRRS located in non-transgenic plant genomic DNA of a second DNAjunction polynucleotide of the second modified transgenic locus; andwherein the method further comprises: (a) contacting the editedtransgenic plant genome with: (i) an RNA dependent DNA endonuclease(RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guideRNA hybridization site of the second OgRRS and the second CgRRS; whereinthe RdDe recognizes a second OgRRS/gRNA and a second CgRRS/gRNAhybridization complex; and, (b) selecting a transgenic plant cell,transgenic plant part, or transgenic plant wherein the second modifiedtransgenic locus or portion thereof flanked by the second OgRRS and thesecond CgRRS has been excised.
 58. The method of claim 54, wherein thetransgenic plant cell is in tissue culture, in a callus culture, a plantpart, or in a whole plant and/or wherein the transgenic plant cell is ahaploid plant cell.
 59. The method of claim 54, wherein the editedtransgenic plant genome is further contacted in step (a) with a donorDNA template, optionally wherein the donor DNA template comprises apolynucleotide sequence comprising non-transgenic plant genomic DNAlocated outside of the modified transgenic DNA which is to be excised,optionally wherein the donor DNA template comprises non-transgenic plantgenomic DNA present in an unmodified plant at the site where thetransgenic locus was originally inserted, and optionally in step (b)selecting a transgenic plant cell, transgenic plant part, or transgenicplant wherein polynucleotides in any of the donor DNA templates haveintegrated in the position in the plant genome where the modifiedtransgenic locus was excised.
 60. A method of obtaining a plant breedingline comprising: (a) crossing two transgenic plants comprising theedited transgenic genomes of any of claims 1 to 11, wherein a firstplant comprising the first modified transgenic locus is crossed to asecond plant comprising the second modified transgenic locus; and, (b)selecting a progeny plant comprising the first and second modifiedtransgenic locus from the cross, thereby obtaining a plant breedingline.
 61. The method of claim 60, wherein the plant breeding line issubjected to a haploid inducer and a haploid plant breeding linecomprising at least the first and second breeding line is selected. 62.A method for obtaining inbred transgenic plant germplasm containingdifferent transgenic traits comprising: (a) introgressing at least afirst transgenic locus and a second transgenic locus into inbredgermplasm to obtain a donor inbred parent plant line comprising thefirst and second transgenic loci, wherein a first OgRRS and a firstCgRRS are operably linked to both DNA junction polynucleotides of atleast the first transgenic locus and optionally wherein a second OgRRSand a second CgRRS are operably linked to the second transgenic locus;(b) contacting the transgenic plant genome of the donor inbred parentplant line with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii)a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridizationsite of the first OgRRS and the second CgRRS; wherein the RdDerecognizes a first OgRRS/gRNA and a first CgRRS/gRNA hybridizationcomplex; and (c) selecting a transgenic plant cell, transgenic plantpart, or transgenic plant comprising an edited transgenic plant genomein the inbred germplasm, wherein the first transgenic locus has beenexcised and the second transgenic locus is present in the inbredgermplasm.
 63. The method of claim 62, wherein the introgressioncomprises crossing germplasm comprising the first and/or secondtransgenic plant locus with the inbred germplasm, selecting progenycomprising the first or second transgenic plant locus, and crossing theselected progeny with the inbred germplasm as a recurrent parent. 64.The method of claim 62, further comprising contacting the transgenicplant genome in step (b) with one or more gene editing molecules thatprovide for excision or inactivation of a selectable marker transgene ofthe second transgenic locus and selecting for a transgenic plant cell,transgenic plant part, or transgenic plant wherein the selectable markertransgene has been excised or inactivated.
 65. The method of claim 62,wherein the gene editing molecules include a donor DNA templatecontaining an expression cassette or coding region which confers auseful trait and the transgenic plant cell, transgenic plant part, ortransgenic plant is selected for integration of the expression cassetteat the site of the selectable marker transgene excision or inactivation.66. The method of claim 62, wherein a second OgRRS and a second CgRRSare operably linked to the second transgenic locus and wherein themethod further comprises contacting the transgenic plant genome of thedonor inbred parent plant line with: (i) an RNA dependent DNAendonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizingto the guide RNA hybridization site of the second OgRRS and the secondCgRRS; wherein the RdDe recognizes a second OgRRS/gRNA and a secondCgRRS/gRNA hybridization complex in step (b); and selecting a transgenicplant cell, transgenic plant part, or transgenic plant wherein thesecond transgenic locus has been excised in step (c).
 67. The method ofclaim 62, wherein the transgenic plant genome is contacted in step (b)by introducing one or more compositions comprising or encoding theRdDe(s) and gRNAs into a transgenic plant cell comprising the transgenicplant genome.
 68. The method of claim 62, wherein the edited transgenicplant genome is contacted in step (a) by introducing one or morecompositions comprising the gRNA into a transgenic plant cell comprisingthe edited transgenic plant genome, wherein the transgenic plant cellcomprises a polynucleotide that encodes the RdDe and the encoded RdDe isexpressed in the transgenic plant cell.
 69. The method of claim 62,wherein the transgenic plant genome is further contacted in step (b)with a donor DNA template molecule comprising an introduced transgeneand a transgenic plant cell comprising an edited transgenic plant genomecomprising an insertion of the introduced transgene in the firsttransgenic locus is selected in step (c).
 70. The method of claim 62,wherein the transgenic plant genome is further contacted in step (b)with: (i) a donor DNA template molecule comprising an introducedtransgene; and (ii) one or more DNA editing molecules which introduce adouble stranded DNA break in the second transgenic locus; and atransgenic plant cell comprising an edited transgenic plant genomecomprising an insertion of the introduced transgene in the secondtransgenic locus is selected in step (b).
 71. The method of claim 62,further comprising: (d) contacting the edited transgenic plant genome inthe selected transgenic plant cell of step (c) with: (i) a donor DNAtemplate molecule comprising an introduced transgene; and (ii) one ormore DNA editing molecules which introduce a double stranded DNA breakin or near the excision site of the first transgenic locus or in thesecond transgenic locus; and, (e) selecting a transgenic plant cell,transgenic plant part, or transgenic plant comprising a further editedtransgenic plant genome comprising an insertion of the introducedtransgene in or near the excision site of the first transgenic locus orin the second transgenic locus.
 72. The method of any one of claims 62to 71, wherein the transgenic plant germplasm is transgenic corn plantgermplasm and wherein the first and/or second modified transgenic locuscomprises at least one modification of a Bt11, DAS-59122-7, DP-4114,GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, MIR604,NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038,MON863, MON87403, MON87403, MON87419, MON87460, MZHG0JG, MZIR098,VCO-Ø1981-5, 98140, or TC1507 original transgenic locus in a transgeniccorn plant genome, wherein the modification comprises the CgRRS in thesecond DNA junction polynucleotide of the first transgenic locus andoptionally the CgRRS in the second DNA junction polynucleotide of thetransgenic locus, and wherein the modification optionally furthercomprises a deletion of at least one selectable marker gene and/ornon-essential DNA in the original transgenic locus.
 73. The method ofany one of claims 62 to 71, wherein the transgenic plant germplasm istransgenic soybean plant germplasm and wherein the first and/or secondmodified transgenic locus comprises a modification of an A5547-127,DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708,MON89788, MST-FGØ72-3, or SYHT0H2 original transgenic locus in atransgenic soybean plant genome, wherein the modification comprises theCgRRS in the second DNA junction polynucleotide of the first transgeniclocus and optionally the CgRRS in the second DNA junction polynucleotideof the transgenic locus, and wherein the modification optionally furthercomprises a deletion of at least one selectable marker gene and/ornon-essential DNA in the original transgenic locus.
 74. The method ofany one of claims 62 to 71, wherein the transgenic plant germplasm istransgenic cotton plant germplasm and wherein the first, second, and/orthird modified transgenic locus comprises at least one modification of aDAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, orMON88913 original transgenic locus in a transgenic cotton plant genome,wherein the modification comprises the CgRRS in the second DNA junctionpolynucleotide of the first transgenic locus and optionally the CgRRS inthe second DNA junction polynucleotide of the transgenic locus, andwherein the modification optionally further comprises a deletion of atleast one selectable marker gene and/or non-essential DNA in theoriginal transgenic locus.
 75. The method of any one of claims 62 to 71,wherein the transgenic plant germplasm is transgenic canola plantgermplasm and wherein the first, second, and or third modifiedtransgenic locus comprises a modification of an GT73, HCN28, MON88302,or MS8 original transgenic locus in a transgenic canola plant genome,wherein the modification comprises the CgRRS in the second DNA junctionpolynucleotide of the first transgenic locus and optionally the CgRRS inthe second DNA junction polynucleotide of the transgenic locus, andwherein the modification optionally further comprises a deletion of atleast one selectable marker gene and/or non-essential DNA in theoriginal transgenic locus.